3. 1... L4,... .n. ‘1» HUI. 14.: mm". ~53“? :- a can...» . 1“ a... (.1 v .. Elf}. war-i. :2; a I} 3 5.... 2.2.»... . :7 llllllllllllllllllllllllllllllllllllllll\HllllHllUl \ 3 1293 01810 (was) 1 LIBRARY Michigan State University This 18 to certify that the dissertation entitled FHA/army,“ DMSEUION or WWW QAPi‘l- 1M TRAA/sc {81 PTloA/AL IN: TIATIOA/ ELONIS’ATION AA/J) REcYcha presented by Let Lei has been accepted towards fulfillment of the requirements for P“ 9' degree in ’BI‘OCMMI'fiTy PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINE return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 1” Wu FUNCTIONAL DISSECTION OF HUMAN RAP74 IN TRANSCRIPTIONAL INITIATION, ELONGATION, AND RECYCLING By Lei Lei A DISSERTATION submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1998 ABSTRACT FUNCTIONAL DISSECTION OF HUMAN RAP74 IN TRANSCRIPTIONAL INITIATION, ELONGATION, AND RECYCLING By Lei Lei Transcription factor 1117 (TFIIF) cooperates with RNA polymerase II (pol 11) during multiple stages of the transcription cycle. Human TFIIF is an 0:182 heterotetramer of RNA polymerase II-gssociating protein 74- and 30-kDa subunits (RAP74 and RAP30). The primary sequence indicates that RAP74 may comprise separate N- and C-terminal domains connected by a flexible loop. Functional data obtained with RAP74 deletion mutants strongly support this model for RAP74 architecture and further show that the N- and C-terminal domains and the central loop of RAP74 have distinct roles during separate phases of the transcription cycle. The N-terminal domain of RAP74 (minimally amino acid 1-172) is sufficient to deliver pol II into a complex formed on the adenovirus major late promoter with the TAT A binding protein, TFIIB, and RAP30. A more complete N—terminal domain fragment (amino acid 1-217) strongly stimulates both initiation and elongation by pol H. The region of RAP74 between aa 136 and 217 is critical for both initiation and elongation, and mutations in this region have similar effects on initiation and elongation. Based on these observations, RAP74 appears to have a similar function in initiation and elongation. The central region and the C-terminal domain of RAP74 do not contribute strongly to single-round initiation or elongation stimulation but do stimulate multiple-round transcription in an extract system. '. 9' N ‘ ding? $39135} U lllL‘iiZC "in; “-8311 The region of RAP74 between a 136 and 217 is highly conserved among divergent organisms including yeast, Drosophila, Xenopus, and human. Because this region is critical for both initiation and elongation, a comprehensive set of point mutations was introduced and mutant proteins were analyzed in transcription assays. Consistent with the analysis of deletion mutants, point mutations in RAP74 also affect both initiation and elongation very similarly, strongly supporting the notion that RAP74 plays an identical role in both initiation and elongation. Several amino acid residues such as L155, W164, N172, I176, and M177 appear to be most critical for the function of RAP74 in initiation and elongation. The lower activities of RAP74 mutants are not due to the inability to bind the transcription complex; in fact, even the most defective mutants have a similar affinity as wild type RAP74 for both the preinitiation complex and the elongation complex. Recent photocrosslinking experiments demonstrate that an adenovirus virus major late promoter template wraps tightly around pol II in a complex containing TBP, TFIIB, TFIIB, TFIIF, and TFIIH. RAP74 is critical for formation of the wrapped structure and the activities of RAP74 mutants in initiation, elongation, and DNA wrapping are very similar. This suggests that RAP74 may induce DNA wrapping in both the preinitiation complex and the elongation complex to facilitate DNA helix untwisting, which is necessary for both initiation and rapid elongation. TFIIF is the only general transcription factor previously shown to stimulate both initiation and elongation by pol 11. Using a sensitive elongation assay, it is demonstrated that TBP, TFIIB, TFIIA, and TFIIE stimulate the elongation activity of TFIIF, possibly by recruiting TFIIF into the elongation complex. To my parents and my wife iv Ma axially Intriques ‘ “Lama's ACKNOWLEDGMENTS I would like to begin by thanking my mentor Dr. Zachary Burton for his generous support, encouragement, patience, and friendship throughout the years. Without his guidance, none of this could have taken place. I thank Drs. Michele F luck, Laurie Kaguni, Lee Kroos, and Steve Triezenberg for their contributions as my committee members. Many thanks to the past and present members of the Burton laboratory. I would especially like to thank Dr. Ann F inkelstein for teaching me various experimental techniques, sharing all the wonderful stories, and making the lab a warm and pleasant place in which to work. I thank Drs. Delin Ren, Ann Finkelstein, Bo Qing Wang, Shawn Chang, Shimin Fang, Stephan Reimers, and Augen Pioszak for valuable reagents and helpful discussions. Delin and Ann have made important contributions to this work. I am grateful to many people in the department for their support and fiiendship. I thank my friends Yuxun Wang, Carla Margulies, Stephan Reimers, and Yuri Nedialkov for their help and encouragement. I thank Drs. Joe Leykam, Kaillathe “Pappan” Padmanabhan, Stephan Reimers, and Kevin Carr for helping with FPLC and computers. I also thank all the members of the Transcription Journal Club for making this weekly event exciting, challenging, and fun. Finally, I thank my parents and my wife for their unconditional love, understanding, and support. MST Ol lISl Ol MST O] C liil’l [UN ( TABLE OF CONTENTS PAGE LIST OF TABLES ......................................................................... x LIST OF FIGURES ......... xi LIST OF ABBREVIATIONS ............................................................ xiii CHAPTER 1 EUKARYOTIC TRANSCRIPTION BY RNA POLYMERASE H ............. 1 General Transcriptional Machinery ............................................. 3 Promoter Architectures ................................................... 3 TATA ............................................................. 3 Inr .................................................................. 4 DPE ................................................................ 5 RNA Polymerase II ....................................................... 6 CTD ................................................................ 8 Holoenzymes ...................................................... 10 General Transcription Factors ............................................ ll TFIID ............................................................... 13 TBP ........................................................ l3 TAFns ...................................................... 14 TFIIA ............................................................... l8 TFIIB ............................................................. 20 TFIIF ................................................................ 22 TFIIB ................................................................ 29 TFIIH ............................................................... 30 Transcriptional Initiation ........................................................... 32 Preinitiation Complex Assembly ......................................... 33 Open Complex Formation ................................................ 37 Abortive Initiation ......................................................... 39 Promoter Escape ............................................................ 39 Transcriptional Elongation ......................................................... 41 Mechanics of Elongation .................................................. 41 Pausing, Arrest, and Termination ........................................ 44 Elongation Factors ......................................................... 45 Transcriptional Termination and Recycling ..................................... 49 MCI] [\lllil R: A Transcriptional Activation ......................................................... 51 Activators ................................................................... 52 Coactivators ................................................................. 54 Mechanisms of Activation ................................................ 6O Transcriptional Repression ......................................................... 66 Overview ............................................................................. 68 CHAPTER 2 FUNCTIONS OF THE N-TERMINAL DOMAIN OF RAP74 IN INITIATION AND ELONGATION .................................................... 72 Introduction ......................................................................... 72 Materials and Methods ............................................................ 75 Transcription factors and extracts ....................................... 75 Construction of RAP74 mutants ........................................ 75 Electrophoretic mobility shift assay .................................... 76 Transcription assays ...................................................... 77 Immobilized templates .......................................... 77 Pulse-spin and pulse-chase single-round assays ............. 77 Elongation stimulation assay ................................... 78 Results ............................................................................... 80 The N-terminal domain of RAP74 supports preinitiation complex assembly .................................................................... 80 The N-terminal domain of RAP74 supports accurate initiation. . 83 The N-terminal domain of RAP74 stimulates elongation by pol II ..................................................................... 90 Discussion ........................................................................... 98 Acknowledgements ................................................................ 102 Clitl’l SUI-D 0f RU [LONG CHIP R‘Xc REGK CHAPTER 3 SITE-DIRETED MUTAGENESIS OF THE N-TERMINAL DOMAIN OF RAP74: CRITICAL AMINO ACID RESIDUES INVOLVED IN ELONGATION ............................................................................ 103 Introduction ......................................................................... 103 Materials and Methods ............................................................ 106 Construction of RAP74 mutants ........................................ 106 Transcription assays ...................................................... 106 Immobilized templates .......................................... 106 Initiation assay: pulse-sarkosyl chase ......................... 106 Elongation stimulation assay: sarkosyl and high salt wash ............................................... 107 Results ............................................................................... 109 Detailed mutagenesis of RAP74 in the critical region betweenaa 136 and 217 .................................................. 109 TFHF mutants affect both initiation and elongation very similarly .............................................................. l 15 Discussion .......................................................................... 127 Acknowledgements ............................................................... 133 CHAPTER 4 FUNCTIONS OF THE C-TERMINAL DOMAIN AND CENTRAL REGION OF RAP74 IN RECYCLING OF RNA POLYMERASE H ......... 134 Introduction ......................................................................... 134 Materials and Methods ............................................................ 136 ' Transcription factors and extracts ....................................... 136 Construction of RAP74 mutants ................. _ ........................ 136 Multiple-round and sarkosyl block assays ............................. 136 G-less cassette pol II trap ................................................ 137 Results ............................................................................... 139 The central region and C-terminal domain of RAP74 stimulate multiple-round transcription ............................................. 139 New initiation resulted from previously unused pol II molecules initiating fi'om previously unused promoters .......................... 150 Discussion ........................................................................... 156 CHIP MC [V EU Hm Rim Acknowledgements ................................................................ CHAPTER 5 FUNCTIONS OF GENERAL TRANSCRIPTION FACTORS IN ELONGATION ....................................................................... Introduction ........................................................................ Materials and Methods ........................................................... Transcription factors and extracts ..................................... Transcription with purified components ............................. Elongation stimulation assay .......................................... Results ............................................................................. RAP74 stimulates transcription fiom a supercoiled template. . TBP, TFIIB, TFIIA, and TF 11E stimulate elongation by pol II. .. TBP, TFIIB, TFIIA, and TFIIE enhance the elongation activities of TFIIF and TFIIF mutants ............................... Discussion ........................................................................ Acknowledgements .............................................................. FUTURE RESEARCH ................................................................. REFERENCES .......................................................................... 162 163 163 165 165 165 166 167 167 167 175 177 181 182 186 labia l. l LIST OF TABLES PAGE CHAPTER 1 Table 1. Human general transcription factors ............................................ 12 .1. I. t- C; l‘ ‘r 1 Iii-u - b P' F'f'”? 51-1316 1 5 figure 3 hrs—1 i are 4 l 5 rrr ._s. i r ('V‘ (I) ' _/I "u 1‘: (TI J.- LIST OF FIGURES PAGE CHAPTER 1 Figure l. A model for the wrapping of the promoter DNA around RNA polymerase during formation of the preinitiation complex ..................................................... 23 Figure 2. Proposed functions of TFIIF in transcriptional initiation and elongation .............................................................. 27 CHAPTER 2 Figure 1. An electrophoretic mobility shift assay was used to analyze the requirement for RAP74 to form DBPolF ............................... 82 Figure 2. Accurate initiation from an adenovirus major late promoter in the HeLa extract is dependent on both RAP3O and RAP74 subunits of TFIIF ..................................................................... 85 Figure 3. Regions of RAP74 required for accurate initiation .................... 87 Figure 4. The region of RAP74 between a 172-217 is critical for both accurate initiation and elongation stimulation .......................... 92 Figure 5. A conserved region of RAP74 between amino acids 170-178 is important for both initiation and elongation ............................ 96 CHAPTER 3 Figure 1. Identification of a region of RAP74 that is critical for transcription initiation and elongation stimulation ..................... 1 l 1 Figure 2. TFIIF mutagenesis ......................................................... 1 14 Figure 3. Analysis of representative TFIIF mutants in elongation .............. l 17 Figure 4. TFIIF containing RAP74(1176A) has a similar affinity to TFIIF wt for elongation complexes but a lower activity .......................... 1 19 Figure 5. The average elongation rates of TFIIF and TFIIF mutants. . . . . . . . 122 Figure 6. TFIIF mutants have very similar activities in accurate initiation and elongation rate stimulation ........................................... 126 xi ligre l. Figue 3. litre 4. Figure 5. Fiat-e 1 Fig.7: 2 5am Figure 7. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 1 Figme 2 Figure 3 A model for the role of TFIIF in isomerization of the elongation complex ........................................................ 131 CHAPTER 4 The C-terminal domain of RAP74 stimulates multiple-round transcription ................................................................ 141 The central region and the C-terminal domain of RAP7 4 cooperate to stimulate multiple-round transcription ............................... 145 RAP74(1-217) is specifically defective for multiple-round transcription ................................................................. 149 Multiple-round transcription in an extract system can be described by a kinetic limitation model ............................................. 153 N- and C-terminal domains of RAP74, which were originally proposed from sequence analysis, correspond to distinct functional domains ......................................................... 158 CHAPTER 5 RAP74 stimulates transcription from a supercoiled template pML(C2AT)A71 ............................................................ 169 General transcription factors stimulate elongation by pol II .......... 17l TBPc, TFIIA, TFIIB, and TFIIE stimulate the elongation activities of TFIIF and TFHF mutants ............................................... 174 AMP ANS All” BSA CKll DNA D18 DPE D11 EDTA EGTA Ric Hi1 HCA PEPES AdMLP AMS ATP bp BSA Da DNA DRB DPE DTT EDTA EGTA FPLC GTP LIST OF ABBREVIATIONS amino acid(s) adenovirus major late promoter ammonium sulfate adenosine triphosphate base pair(s) bovine serum albumin casein kinase II carboxyl terminal domain of the largest subunit of RNA polymerase II cytidine triphosphate Dalton deoxyribonucleic acid 5,6-dichlorobenzimidazole riboside downstream promoter element dithiothreitol ethylenediamine tetraacetic acid ethyleneglycol-bis-(B-aminoethyl ether) N, N, N’, N’-tet_raacetic acid fast protein liquid chromatography guanosine triphosphate histone acetyltransferase hydrophobic cluster analysis histone deacetylase N-(2-hydroxyethyl)piperazine-N’-(2—ethanesulfonic acid) xiii NT? PAG PCR PBS kb mRNA at PAGE PCR PBS pol I polII polIII rRNA sarkosyl SDS TAFn TATA TBP TFII tRNA Initiator element lcilobase pairs kilodaltons messenger RNA nuclear magnetic resonance nucleotide(s) nucleoside triphosphate polyacrylamide gel electrophoresis polymerase chain reaction phosphate buffered saline RNA polymerase I RNA polymerase II RNA polymerase 1H RNA polymerase II associating protein ribonucleic acid ribosomal RNA N-lauroylsarcosine sodium salt sodium dodecyl sulfate TBP associated factor of RNA polymerase H transcription TATA element or TATA box ‘ TATA box-binding protein transcription factor of RNA polymerase II transfer RNA xiv LTP Amino Acid ( Alum: lime imagine mm Acid Cysteine Glam Acii Glutamim Glycine Hbfidne talcum Wine [line Mionine thilalanin Mme Settle Theme Ilipzophan Tims’me Value UTP Uridine triphosphate Amino Acid Codes Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine He 1 Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Tr DNAdep l) saints message Hi (pol H 1168’ re RNA pol 1‘ Wm. 198:3). 1 Wide ' CHAPTER 1 TRANSCRIPTION BY EUKARYOTIC RNA POLYNIERASE II Transcription of nuclear genes in eukaryotes is carried out by three different DNA-dependent RNA polymerases (reviewed in Young, 1991). RNA polymerase I (pol I) synthesizes the ribosomal RNA (rRNA); RNA polymerase H (pol II) synthesizes the messenger RNA (mRNA) and some small nuclear RNA (snRNA); and RNA polymerase III (pol III) synthesizes the SS rRNA, transfer RNA (tRNA), and some snRNA. Each of these three eukaryotic RNA polymerases is composed of 8-14 polypeptides. All three RNA polymerases require the assistance of accessory transcription factors for specific transcription initiation. The complexity of the eukaryotic RNA polymerases and their transcription factors likely reflects the need for elaborate controls on transcription in eukaryotes. When transcribing mRNA-encoding genes, pol II alone is not sufficient for specific initiation (Roeder, 1976; Weil et al., 197 9). An additional set of protein factors is required for accurate initiation fi'om a promoter DNA sequence in vitro (Matsui et al., 1980). These accessory factors are termed GTFs (general transcription factors) and include TBP (TATA-binding protein) or TFIID, TFIIA, TFIIB, TFIIF, TFIIE and TFIIH (“'I‘F” for transcription factor, and “II” for RNA polymerase H) (reviewed in Conaway and Conaway, 1993; Zawel and Reinberg, 1993; Roeder, 1996; Orphanides et al., 1996; Hampsey, 1998). The entire set of GTFs is composed of about 30 polypeptides and most of the corresponding cDNAs have been isolated. Similar factors have been identified in human, yeast, rat and Drosophila systems. These GTFs along with pol II are often referred to as transcription Aldrc from naked I responsive tr transcription The pacing Pamjape er Manpdor Mason 13‘ “passion ( 5880911088 (: “filth bridg ”Wilmer The mm“ m “We at msmptior Milling-y z mmpfioj Rhea} mar .3316 allies referred to as the general transcriptional machinery to emphasize their importance in transcription. Although pol II and the GTFs are sufficient for accurate initiation of transcription from naked DNA templates in vitro, this transcription is neither dependent on nor responsive to the presence of transcriptional activators and is therefore known as “basal” transcription. Transcription in an eukaryotic cell, however, is a tightly regulated process. The packaging of DNA into chromatin results in global gene repression (reviewed in Paranjape et al., 1994). Moreover, sequence-specific DNA binding proteins regulate the transcription in a gene-specific manner (Mitchell and Tjian, 1989). Transcriptional repressors bind to specific DNA sequences (repressor-binding sites) and inhibit the expression of target genes, while transcriptional activators bind to specific DNA sequences (activator-binding sites) and induce the expression of target genes. Cofactors, which bridge the interaction between a DNA-bound regulator and the general transcriptional machinery, sometimes are also required for gene activation or repression. The overall outcome of gene induction is therefore a combined result of “anti-repression” and “true activation” (Roeder, 1991; Orphanides et al., 1996). Although the “basal” transcription may not occur in vivo, the components of the general transcriptional machinery and the process of basal transcription are nevertheless the ultimate targets of transcriptional regulators. Studies of the basic mechanisms of transcription and the general machinery not only improve our understanding of the. fundamental concepts of gene expression, but also provide us with the fiamework necessary to understand how regulators work. In t] he oonpor manpne gircn Alt asaguider GDIRA] Promoter . The tgdatory e mhly 0' Selecting < initiator SCq 53‘? a TAT inseam 8731110135. W‘lfic SEQ ElkJCh reg-,1} lAIA TAT StaWeir” “Bears are In this chapter I will review the basics of mRNA transcription by pol 11, including the components of general transcriptional machinery and the process of basal transcription. A brief summary of transcriptional activation and repression will also be given. At the end, an overview will be given to reemphasize the basic fimction of TFIIF as a guide to the following chapters. GENERAL TRANSCRIPTION AL MACHINERY Promoter Architectures The promoter of an mRNA-encoding gene can be divided into core elements and regulatory elements (reviewed in Harnpsey, 1998). The core elements define the site for assembly of the preinitiation complex (PIC) and contribute to both the strength and selectivity of a promoter. The core elements include a TATA sequence (TATA), an initiator sequence (Inr), and a downstream promoter element (DPE). Arpromoter can have a TATA box, an Inr sequence, or both a TATA and an Inr. DPE appears to function in conjunction with the Inr element as a TFIID binding site at some TATA-less promoters. Regulatory elements, generally located outside the core promoter, are gene- specific sequences that serve as binding sites for transcriptional activators or repressors which regulate the level of transcription. TATA TATA elements are located about 30 base pairs (bp) upstream of the transcription start site in most eukaryotes except in yeast Saccharomyces cerevisiae where TATA elements are typically 40 to 120 bp upstream of the start site (Hampsey, 1998). The menus s 5th 1991 with reduce binding prc The binding the (Hoop Pro: been iden’n dconnucle (“his and l dmdmt Irii'rely to [3555 (T8 TATA [o 11‘ consensus sequence for TATA elements is TATAAA (Singer et al., 1990; Wobbe and Struhl, 1990). Many derivatives of this sequence also confer TAT A function, although with reduced activity. The TATA sequence serves as the binding site for the TATA binding protein (TBP). The TBP-TATA association nucleates the assembly of the PIC. The binding of TBP is a step that can be rate limiting for initiation both in vitro and in vivo (Hoopes et al., 1992; Klein and Struhl, 1994). Promoters lacking canonical TATA elements (TATA-less promoters) have also been identified, especially in many housekeeping genes such as the terminal deoxynucleotidyl transferase (T (11') gene and the dihydrofolate reductase (DHFR) gene (W eis and Reinberg, 1992). Transcription from TATA-less promoters remains TBP- dependent, although the rate-limiting step in PIC assembly at TATA-less promoters is unlikely to be TBP recruitment. Other components of the general machinery, such as the TAFns (TBP-associated factors in TFIID), may recognize promoter elements other than TATA to nucleate the PIC assembly on the TATA-less promoters. Initiator elements are DNA sequences encompassing the transcription start site. The Inr was first identified at the TATA-less promoter of the mammalian TdT gene and subsequently found at many promoters, either TATA-containing or TATA-less (W eis and Reinberg, 1992). It now appears that most promoters contain an Inr, although the nucleotide sequence of this element is not highly conserved. Proteins that bind Inr elements include TFII-I, YYl, CIF150, E2F, USF, as well as pol 11 itself (reviewed in Smale, 1997; Weis and Reinberg, 1992). Genetic evidence in yeast indicated that TFIIB lAngS down banning them ii Therein m addi {Mini-'15- may also interact with the Inr in determining the start site (Pinto et al., 1992; Pinto et al., 1994). TAFns are also implicated in recognizing the Inr to function as promoter selective factors. Among these Inr-binding proteins, CIFISO is a homolog of Drosophila TAFn150 (dTAFn150), which binds promoter DNA overlapping the Inr region (Kaufmann et al., 1996; Kaufinann et al., 1998; Verrijzer and Tjian, 1996). A recombinant TBP-TAFHISO- TAF 11250 subcomplex is minimally required for efficient utilization of Inr and the downstream promoter element (V errijzer et al., 1994; Verrijzer et al., 1995). A human homolog of dTAF [1150, named human TAFn150 (hTAFu150), was recently identified as a bona fide TFIID subunit and found to be homologous to CIF150 (Martinez et al., 1998). Therefore, the fimction of recognizing the Inr by TAFu subunits is conserved. It appears that additional factors are essential for transcription from TATA-less promoters (Martinez et al., 1998). The functions and mechanisms of other Inr-binding proteins remain to be further determined. It is possible that different Inr-binding proteins are utilized at different promoters. DPE The downstream promoter element (DPE) was initially identified in Drosophila and is located about 30 bp downstream of the start site (Burke and Kadonaga, 1996; Burke and Kadonaga, 1997). The DPE works in conjunction with the Inr element as a TFIID binding site at some TATA-less promoters. The historic-like TAFns including dTAFu60 and dTAFn 40 interact specifically with the DPE. It is interesting to note that some TAFns also contact the DNA about 30 bp downstream of the start site on the TATA-containing adenovirus major late promoter (V errijzer et al., 1995; Oelgeschlager ad, 1996). the TATA se the promoter fitters (Rob RNA POLl Bast spenfidty f mints. P cams of ‘. is masks For cramp; 5851(3ch “he‘s-wt Mia] cc “'5 prr 1 R957. and 0m’RPB4 1h et al., 1996). One explanation is that the PIC generally contacts DNA fiom upstream of the TATA sequence all the way to about 30 bp downstream of the start site. In doing so, the promoter DNA is fully wrapped around the polymerase and general transcription factors (Robert et al., 1998). RNA POLYMERASE H Bacterial RNA polymerase consists of an 0288’ core enzyme and a a promoter specificity factor. All three eukaryotic nuclear RNA polymerases are also multi-subunit enzymes. Pol II is generally composed of 10-12 subunits; for example, yeast pol H consists of 12 polypeptides encoded by the RPBI to RPBIZ genes (Young, 1991). There is extensive structural conservation among the pol II subunits from diverse organisms. For example, six subunits of human pol H can functionally replace their homologs in yeast (McKune et al., 1995). Eukaryotic RNA polymerases consist of common as well as class-specific subunits. Five subtmits, pr5, pr6, and pr8, pr10, and pr12, are essential components of all three eukaryotic RNA polymerases. The prl, pr2, pr3, and pr11 subunits of pol H are homologous to subunits of pol I and pol 1H. Only pr4, pr7, and pr9 are unique to pol H. Among the genes encoding yeast pol H subunits, only RPB4 and RPB9 are not esential for cell viability. The three largest pol H subunits are related to the bacterial core RNA polymerase subunits and are largely responsible for RNA catalysis (Young, 1991). The largest pol H subunits, prl, is homologous to the [3’ subunit of bacterial RNA polymerase. The second largest pol H subunit, pr2, is homologous to the [3 subunit of bacterial RNA polymerase. pr3 is somewhat related to the a submit of bacterial RNA polymerase. No mhuni No subunit of pol H appears to be closely related to the bacteria] 0' subunit. The bacterial polymerase active site is made up of a modular arrangement of B’ and B subunits, suggesting that prl and pr2 may constitute the active site of pol H (Mustaev et al., 1997; Severinov et al., 1996; Zaychikov et al., 1996; Markovtsov et al., 1996). prl and B’ are involved in template DNA binding, while pr2 and B bind nucleotide substrates. Yeast genetics provide some clues regarding the in vivo functions of individual pol H subunits. Different mutations in RPBl and RPB2 have been found to affect either the start site selection or transcriptional elongation, demonstrating that both prI and pr2 are important in both initiation and elongation (Berroteran et al., 1994 Hekrnatpanah and Young, 1991; Archambault et al., 1992; Powell and Reines, 1996). pr3 is involved in pol H assembly, a similar function as the bacterial a subunit (Young, 1991; Kolodziej and Young, 1991; Kolodziej and Young, 1989; Kolodziej et al., 1990). pr9 is implicated in defining the start site, perhaps through interaction with TFHB (Hull et al., 1995; Sun et al., 1996). Both pr9 and TFHB have a zinc ribbon motif which may be involved in start site selection. Biochemical analyses indicate that pr4 and pr7 are loosely associated with pol H and a form of yeast pol H without pr4 and pr7 is active in elongation in vitro (Edwards et al., 1991). However, both subunits are required for accmate initiation in vitro. The functions of other pol H subunits remain unclear. The protein envelope structure of various RNA polymerases has been determined by two-dimensional crystallography of microcrystalline arrays (Polyakov et al., 1995; Darst et al., 1939; Asturias et al., 1997; Darst et al., 1991). Prominent features ofthese structures are finger-like projections in the RNA polymerase that close to form a channel large enough to accommodate the DNA template. For E. coli RNA polymerase, this predated abreast presence 0 1997; Day. channel is open in the initiating holoenzyme and closed in the elongating enzyme. This suggests that the DNA penetrates through the channel, which closes around the template during elongation to prevent termination (Polyakov et al., 1995; Darst et al., 1989). The structure of an RNA polymerase can be described as a “hand” with a “palm”, “thumb”, and “finger”. The DNA template is suggested to run across the palm and throughThe channel, grasped by the thumb and finger. The active site for RNA synthesis is postulated to be close to the channel between the thumb and the finger. The yeast RNA polymerase H can display the thumb and finger either apart or together, depending on the presence of pol H subunits pr4 and pr7 and TFIIE in the complex (Asturias et al., 1997; Darst et al., 1991). Photocrosslinking experiments suggest that the DNA template is wrapped around the polymerase and general transcription factors in the pol II preinitiation complex (Robert et al., 1998; Forget et al., 1997; Kim et al., 1997). The DNA wrapping is proposed to play an important role in the formation of open complex during initiation and the advance of the transcription “bubble” during elongation (Lei et al., 1998; Robert et al., 1998). A unique feature of pol H is the presence of tandem repeats of a heptapeptide sequence at the carboxy-terminus of its largest subunit (reviewed in Young, 1991; Dahmus, 1996). The carboxy-terminal repeat domain (CTD).has a consensus sequence YSPTSPS and is highly conserved among eukaryotic organisms. The repeat length appears to increase with increasing genome complexity, for example, there are 26 or 27 phasphon lherefore phosphor hmd‘ CTD. an the PIC, ' Consent hinatior Who 1993; Se IWSC ‘. mbede repeatsinyeastpol H, dependingonthestrain, and 52 repeatsinhumanpolH (Young, 1991). The CTD is highly phosphorylated on the two SP serines in vivo. It can also be phosphorylated on the tyrosine residues (Dahmus, 1994; Dahmus, 1995; Dahmus, 1996). Therefore, because of the repetitive sequences, about 100 to 150 potential phosphorylation sites are present within the CTD of human pol H. There are at least two forms of RNA pol H in vivo, designated pol HO, which is hyperphosphorylated at the CTD, and pol HA, which is hypophosphorylated. The pol HA form preferentially enters the PIC, whereas pol H0 is found in the elongating complex (Dahmus, 1996). Conversion of pol HA to pol HO occurs concomitant or shortly after the transition from initiation to elongation (Lu et al., 1991; O. Brien et al., 1994). The CTD can be phosphorylated by many kinases in vitro, including TFIH-I (Feaver et al., 1991; Lu et al., 1992; Serizawa et al., 1992), P-TEFb (Marshall et al., 1996), SrblO/Srbll (Liao et al., 1995), Cdc2 (Cisek and Corden, 1989), and Ctkl (Lee and Greenleaf, 1991). It remains to be determined whether these CTD kinases are gene specific or if they affect different steps during the transcription cycle. A phosphatase activity specific for dephosphorylation of the CTD has also been identified (Chambers and Dahmus, 1994). This CTD phosphatase activity is regulated by TFIIF and TFHB (Chambers et al., 1995). l The RAP74 subunit of TFHF stimulates CTD phosphatase activity while TFIIB inhibits this stimulatory effect of TFHF. Because pol HA preferentially enters the PIC, the CTD phosphatase, TFHF, and TFHB appear to interact to regulate pol H recycling (Lei et al., 1998). Althou mirely Clair. 00nt pro DHFR promot sensation of n 1998’). This rr Beside mRNA proces Mcfneken et mfdkaryotes CTD (Stem Holoenlme, RNA him, Suggest 1939; resign. bi the idem elk- In .. SR3 mediate 1994)- Burn Although the CTD is essential for cell viability, its function in transcription is not entirely clear. RNA pol H lacking the CTD is able to initiate transcription fiom TATA- eontaining promoters in vitro, although not from TATA-less promoters such as the DHFR promoter (Akoulitchev et al., 1995; Buermeyer et al., 1995). Moreover, the activation of many yeast genes is independent of the TFIH-I kinase in vivo (Lee and Lis, 1998). This may suggest a functional redundancy among various CTD kinases. Besides its frmctions in transcription, the CTD has also been implicated in pre- mRNA processing such as splicing, 5’ capping and 3 ’-end formation (Greenleaf, 1993; McCrackm et al., 1997a; McCracken et al., 1997b; Yue et al., 1997). This suggests that in eukaryotes the mRNA transcription and processing steps may be coupled through the CTD (Steinmetz, 1997). Holoenzymes RNA pol H and the GTFs can assemble in a defined order on promoter DNA in vitro, suggesting stepwise assembly of the PIC (Van Dyke et al., 1988; Buratowski et al., 1989; reviewed in Zawel and Reinberg, 1993). However, this model has been challenged by the identification of RNA polymerase H holoenzymes fi'om yeast and mammalian cells. The yeast pol H holoenzyme contains all the subunits of pol H (core pol II), an SRB/mediator complex, and a subset of GTFs (Koleske and Young, 1994; Kim et al., 1994). Unlike the core pol H, the holoenzyme supports activated transcription when supplemented with only TBP (instead of TFHD) and the remaining GTFs in reconstituted transcription systems. This property is attributed to the SRB/mediator complex that contains coactivators that physically link activators to the basal transcription machinery. 10 This fern helm)“ and it in; Wade et hat the (Chan et er al., 19* Kirstin has an GENE}; 1 from but insane. hOi‘ihan Conan-a; is in 111 cells This form of pol H holoenzyme is often referred to as the SRB/mediator-containing holoenzyme. Another distinct form of yeast pol H holoenzyme has also been discovered and it appears to be less abundant and regulates only a subset of genes (Shi et al., 1997; Wade et al., 1996; Shi et al., 1996). Several forms of mammalian pol H holoenzymes have also been isolated through either immunoprecipitation or affinity chromatography (Chao et al., 1996; Maldonado et al., 1996b; Ossipow et al., 1995; Scully et al., 1997; Pan et al., 1997). These holoenzymes differ in their composition but all respond to transcriptional activators. The identification of different pol H holoenzymes fiorn both yeast and human suggests that the assembly of a pol H holoenzyme may be a dynamic and complex process and different forms of pol H holoenzymes may coexist inside cells. GENERAL TRANSCRIPTION FACTORS The GTFs include TFHD, HA, IIB, HF, HE and IH-I and were identified initially fi'om human HeLa cell extracts as protein factors necessary for accurate transcription initiation by the purified RNA pol H from double-stranded DNA templates in vitro (Orphanides et al., 1996; Roeder, 1996; Hampsey, 1998; Conaway and Conaway, 1993; Conaway and Conaway, 1997). The compositions and major properties of human GTFs are summarized in Table 1. Similar factors were also identified in yeast, Drosophila and rat cells, and the GTFs isolated from divergent organisms are highly conserved. ll Table 1. Human general transcription factors Factors Subunits Characteristics TFIH) TBP Binds TATA element, contacts TFIIB and TFIIA, nucleates PIC assembly; required for pol I, pol II, and pol 1H transcription TAFHZSO Scaffold for TFIH) assembly, binds TBP and inhibits TBP- TATA interaction; cell cycle progression (GI/S), serine kinase (RAP74), HAT TAFnlSO, CIFISO Contacts Inr TAF "100/ 95 W40 repeats TAFn80/7O Histone H4 similarity, Drosophila homolog (dTAFn60) contacts DPE TAFHSS TAF "32/31 Histone H3 similarity, Drosophila homolog (dTAFu40) contacts DPE TAFu3O Binds estrogen receptor TAF"28 Histone fold TAFHZO/ 15 Histone H2B similarity TAF "18 Histone fold TFIIA TFHAa —“ Both a and B are derived from one gene, likely TFIIAB posttranslationally processed; binds TBP and stabilizes TBP- TFIIAy _ TATA interaction TFIIB TFIIB Binds TBP and stabilizes TBP-TATA interaction, recruits pol II/TFIIF, start site selection; zinc ribbon, cyclin repeats, conformational change TFIIF RAP30 0' homology, cryptic DNA-binding domain (helix-turn-helix); pol 11 delivery, DNA wrapping, initiation and elongation RAP74 Pol H delivery, DNA wrapping, initiation, elongation, and reinitiation; interacts with TFHB, stimulates CTD phosphorylation; phosphorylated in vivo, can be phosphorylated in vitro by CKH, TAFHZSO, and TFlH-I kinases TFHE TFIIEa _ Recruits TFIH-I and stimulates TFlH-I activities (DNA helicases TFHEB _. and CTD kinase); promoter melting, DNA wrapping TFIIH ERCC3, XPB 3’-5 ’ helicase, required for both transcription and NER ERCC2, XPD 5’-3 ’ helicase, required for NER but not transcription p62 NER p52 NER hSSLl NER, zinc-binding cdk7, M015 CTD kinase, component of CAK cyclin H cdk7 partner, component of CAK MAT-l Zinc-binding, CAK assembly factor p34 Zinc-binding PIC - preinitiation complex; HAT - histone acetyltransferase; Inr - initiator element; DPE - downstream promoter element; CTD — the C-terminal domain of RNA polymerase II; CKH - casein kinase H; NER - nucleotide excision repair; CAK - cdk7-activating kinase (cdk7, cyclin H, and MAT-l) 12 TFIID TF1 ind TBP-a; 1996). The initiation c hmitng on affect thel TBP TFIID . T'FIH) is a multisubunit complex consisting of the TATA binding protein (TBP) and TBP-associated-factors (T AF 11$) (reviewed in Hernandez, 1993; Burley and Roeder, 1996). The binding of TBP or TFHD to a promoter is the first step in assembling an initiation complex (Nakajima et al., 1988; Zawel and Reinberg, 1993). This step is rate- limiting on many promoters both in vitro and in vivo and many regulators appear to affect the TBP-TATA interaction. TBP is a universal transcription factor, required for initiation by all three eukaryotic RNA polymerases (Cormack and Struhl, 1992; Hernandez, 1993; White and Jackson, 1992). In each case, TBP is associated with a set of other polypeptides. Besides being a subunit of TFHD, TBP was also identified as an essential component of pol I transcription factor SL1 and pol HI transcription factor TFHIB (Comai et al., 1992; Kassavetis et al., 1992; Lobo et al., 1992; Taggart et al., 1992). TBP also associates with SNAPc, a protein complex required for transcription of certain snRNA genes by pol H or pol IH (Hernandez, 1993). TBP has been isolated from various eukaryotic organisms and it ranges in molecular weight fi'om 22 kDa (Arabidopsis) to 38 kDa (human and Drosophila) (reviewed in Burley and Roeder, 1996). TBP can be divided into two structural domains, a highly conserved C-terminal domain and a more divergent N-terminal domain. The conserved C-terminal domain of TBP (TBPc) consists of two imperfect direct repeats. TBPc is suficient to bind the TATA sequence and direct specific transcription initiation l3 iniitm whet Honhoshi et for formant) terminal dor regimes p0 The novel DNA (Nikolas et binding sur Whining: other DNA clement L the DNA a‘ path of the Such as If and GTFs. Initiation E in vitro when supplemented with the remaining GTFs and pol H (Hoey et al., 1990; Horikoshi et al., 1990; Peterson et al., 1990; Zhou et al., 1993). TBPc is also sufficient for formation of the complete TFIH) complex (Zhou et al., 1993). Although the N- terminal domain is divergent, it is conserved among vertebrate forms of TBP and regulates pol HI transcription at the U6 promoter (Mittal and Hernandez, 1997). The crystallographic structures of TBP and TBP-TATA complexes revealed a novel DNA binding fold, resembling a molecular “saddle” that sits astride the DNA (Nikolov et al., 1992; Kim et al., 1993; Kim et al., 1993; Nikolov et al., 1996). The DNA binding surface is a concave antiparallel B-sheet and the convex seat of the saddle consisting of four tit-helices is available for interaction with other factors. Unlike many other DNA-binding proteins, TBP recognizes the minor groove of the 6-bp TATA element. Upon binding, TBP induces kinks at both ends of the TATA element and bends the DNA about 80° toward the major groove. The bending causes a severe change in the path of the DNA and increases the proximity of proteins bound on either side of TBP, such as TFHA and TFHB. This could initiate wrapping of promoter DNA around pol H and GTFs. The wrapping of DNA is suggested to be important for both transcriptional initiation and elongation (Robert et al., 1998; Lei et al., 1998). TAFns In addition to TBP, TFHD contains 8 to 11 additional TBP-associated factors (T AF us) ranging in size fi'om 15 to 250 kDa (Goodrich and Tjian, 1994a; Burley and Roeder, 1996; Hampsey, 1998). TAFns have been identified from human, Drosophila, and yeast, and there is significant conservation among most TAFn subimits isolated fiom 14 (him or: 1.85130 (t TBP and e2 complexes sihinit of (hill N hlAlngl ylAFuH. E2 the TAT! SFlesh la different organisms. Human TAFn250 (hTAF 11250) and its counterparts, Drosophila T AFn230 (dTAFn230) and yeast TAFn145 (yTAFn145, also known as yTAFn130), bind TBP and each is believed to be a scaffold for the assembly of other TAFus into TFIID complexes. hTAF 11250 is a bipartite protein kinase with specificity for the RAP74 subunit of TFHF (Dikstein et al., 1996). hTAFn250 is also a histone acetyltransferase (HAT) (Mizzen et al., 1996). Both the kinase domains and the HAT domain of hTAFn250 are conserved in dTAFn230, but only the HAT domain is conserved in yT‘AFu145. Earlier studies suggested that TFHD has a weaker affinity than TBP for binding the TATA element (Nakatani et al., 1990; Aso et al., 1994). Studies with a cell-fiee system lacking TAFns revealed that TAFns impair fimctional PIC formation (Oelgeschlager et al., 1998). Investigations using partially reconstituted TFHD complexes showed that hTAF 11250 represses the basal transcription activity of TBP (Verrijzer et al., 1995; Guermah et al., 1998). It was shown that hTAFn250, dTAFu230, and yTAFn145 each inhibit TBP binding to the TATA box in vitro (Kokubo et al., 1993; Kokubo et al., 1998; Nishikawa et al., 1997). Mutagenic analyses indicated that dTAF 11230 binds both the convex side and the concave DNA binding surface of TBP (Kokubo et al., 1994; Nishikawa et al., 1997). Transcriptional activator VP16 binds competitively with dTAF 11230 to the DNA-binding domain of TBP (Nishikawa et al., 1997). The negative regulation of transcription within the TFHD multisubtmit complex was further supported by the three-dimensional structure of a TBP-dTAFn230 complex. This NMR structure of a protein complex consisting of the core domain of yeast TBP (mnino acids 49-240) and the N-terminal region of dTAFn230 (amino acids 11-77) 15 confirm the cone upon in! inwoun resin c of 181’. within shine Trim confirmed that the N-terminal fragment of dTAF 11230 interacts directly and tightly with the concave DNA binding surface of TBP (Liu et al., 1998). This region of dTAF 11230, upon interacting with TBP, undergoes a conformational change and mimics the partially unwound structure of the TATA DNA sequence. It is unlikely that both the N-terminal region of dTAFn230 and the TATA element can occupy the same DNA-binding surface of TBP. These evidences suggest that, prior to the TBP-TATA interaction, a conformational change within the TFHD complex must occur to release the DNA binding surface of TBP fi'om interacting with other TAF 11 subunits including dTAFn230. Transcriptional activators, such as VP 1 6, may facilitate this process. Biochemical and crystallographic analyses indicate that a histone octamer-like structure may exist within TFIH) (Hoffinann et al., 1997 ; Hof‘finann et al., 1996). I-Iistone H3-, H4-, and H2B-like TAFns have been identified in human, Drosophila, and yeast TFIH) complexes (Btn'ley and Roeder, 1996; Xie et al., 1996; Nakatani et al., 1996). In the case of human TFHD, a putative hTAFn histone octamer-like structure would contain a heterodimer of hTAFn31 (histone H3-1ike) and hTAFn80 (H4-like) sandwiched between two hTAFn20 (HZB-like) homodimers. No H2A—like TAFn has been identified. This octamer-like structure may wrap DNA on the surface of TFIH). This would explain the extended DNase I footprint and photocrosslinking of TFIH) compared with those of TBP on many promoters (V enijzer et al., 1995; Oelgeschlager et al., 1996; Burke and Kadonaga, 1996). However, it was recently discovered that the promoter DNA in the preinitiation complex (PIC) containing pol H, TBP, HA, HB, HE, HF and IH-I is wrapped in a similar fashion as that in the DNA-TFIID complex (Robert et al., 1998). Because these structures occupy the same core promoter region, they can not 16 whhp eccentric DNA eor mnmn seen mend heme Brie at with 00: manta require Shims “61.1! $11wa both be present simultaneously. TAFns must move within the TFHD-DNA complex to accommodate assembly of the PIC. It is intriguing to note that no formation of a protein- DNA complex containing TFIH), the remaining GTFs, and pol H has been reported. TAFns appear to play multiple roles in transcription. First, TAFns can function as promoter selectivity factors (Smale et al., 1990; Pugh and Tjian, 1991; Zhou et al., 1992; Martinez et al., 1994; Verrijzer and Tjian, 1996). TAFus are important in recognizing core promoter elements including both the Inr and DPE (Kaufmann and Smale, 1994; Purnell et al., 1994; Verrijzer et al., 1994; Verrijzer et al., 1995; Hansen and Tjian, 1995; Burke and Kadonaga, 1996; Burke and Kadonaga, 1997). Promoter hybrid experiments with conditional TAFu mutants in yeast demonstrate that the requirement for TAFu14S maps to core promoter elements (Shen and Green, 1997). Second, TAFns can function as coactivators (V errijzer and Tjian, 1996). TAFns were previously shown to be generally required for activator-dependent transcriptional stimulation in human and Drosophila systems in vitro (Pugh and Tjian, 1990; Dynlacht et al., 1991; Tanese et al., 1991; Chen et al., 1994; Jacq et al., 1994; Chiang and Roeder, 1995). This paradigm was challenged by the observations that yeast TAFns are only required for transcriptional activation of a subset of genes in vivo (Apone et al., 1996; Moqtaderi et al., 1996; Walker et al., 1996). The TAF 11 firnction at these promoters is associated with core promoter elements instead of elements bound by activators (Apone et al., 1996; Moqtaderi et al., 1996; Walker et al., 1997). Fmthermore, the yeast SRB/mediator-containing pol H holoenzyme is capable of responding to activators in the absence of TAFns in vitro (Koleske and Young, 1994; Kim et al., 1994). It was recently reported that transcriptional activation can occur independent of TAFns in a highly pmified in vitro system, although this TBP-mediated l7 mat obsen': regent sing a stem al.,. 199 filed; . "its TATA bl defined a activation can be stimulated by the addition of TAFns (Wu et al., 1998). The earlier observations that TAFns were necessary for activation are attributed to the presence of negative factors in those systems. The TAFu-independent activation was also observed using a HeLa nuclear extract depleted of the major TAFns by immunoprecipitation (Oelgeschlager et al., 1998). It was shown that the TAFn-independent activation occurs predominantly at the level of productive preinitiation complex assembly. The addition of TAFns actually inhibits the formation of PIC. Human homologs of SRB proteins are implicated in mediating the transcriptional activation in the absence of TAFus (Oelgeschlager et al., 1998). Third, TAFns can function as repressors in a reconstituted system in the absence of activators (Verrijzer et al., 1995; Guermah et al., 1998; Malik et al., 1998). The addition of an activator and a coactivator can relieve this inhibition (Malik et al., 1998). The inhibitory effect of TAFns could be caused by the TAF 11250- TBP interaction which inhibits the binding of TBP to the TATA element (Liu et al., 1998). It could also be caused by the formation of a wrapped TFHD-promoter structure which precludes the formation a functional PIC (Oelgeschlager et al., 1996; Robert et al., 1998). Transcriptional activators and coactivators may relieve the inhibitory effects of TAFus by removing hTAF 11250 from TBP to facilitate the TBP-TATA interaction and displacing the extensive contacts between TAFns and the core promoter. TFHA TFHA associates with the PIC through interaction with TBP and stabilizes TBP- TATA binding (Buratowski et al., 1989; Imbalzano et al., 1994). Although initially defined as a GTF, TFHA is dispensable for accurate initiation in a purified system 18 Reed I . lea: Er: directed by TBP (Matsui et al., 1980; Reinberg et al., 1987; Sayre et al., 1992; Sun et al., 1994; Yokomori et al., 1994). However, TF HA plays important roles in transcriptional activation in vitro. First, TFHA mediates displacement of transcriptional repressors such as Drl-DRAPl/N C2, PC3/Dr2, HMGl, and Motl fiom the TFIH) complex, a process known as “antirepression” (Auble et al., 1994; Ge and Roeder, 1994a; Inostroza et al., 1992; Meisterernst et al., 1991; Merino et al., 1993). Second, TFHA functions in “true activation” by interacting with specific transcriptional activators and coactivators (Ozer et al., 1994; Yokomori et al., 1994; Yokomori et al., 1993; Ge and Roeder, 1994a; Shykind et al., 1995). TFHA is required to overcome a rate-limiting step during formation of the open transcription complex (Wang et al., 1992). cDNAs encoding TFHA subunits have been isolated from human (DeJong and Roeder, 1993; DeJong et al., 1995; Ma et al., 1993; Ozer et al., 1994; Sun et al., 1994), yeast (Ranish et al., 1992), and Drosophila (Y okomori et al., 1993; Yokomori et al., 1994). Human and Drosophila TFHA consist of three subunits with molecular masses of 37 (or subunit), 19 (B subunit) and 13 kDa (7 subunit). or and B subunits are encoded by a single gene and appear to be produced by a protein processing event. Yeast TF HA contains two subunits with molecular masses of 32 and 13 kDa, encoded by TOAI and TOA2 genes respectively. The 32 kDa TOAl gene product is homologous to the human (1 subunit at its amino terminus and the human B subunit at its carboxyl terminus. The 13 kDa TOA2 product is homologous to the human 7 subunit. Crystal structures of yeast TFHA-TBP-TATA complexes have been solved (Geiger et al., 1996; Tan et al., 1996). The TOAl and TOA2 polypeptides interact extensively in the complexes. The carboxyl termini of TOAl and TOA2 form a compact l9 hsheet st form 3 f0 and with intends forn-heli minip nechani: in entire} 1996'). I htrnin 1 he {ism form [he B-sheet structure termed B sandwich or B barrel. The amino termini of TOAl and TOA2 form a four-helix btmdle. TFHA interacts with the amino-terminal region of core TBP and with DNA upstream of the TATA element. The B-sandwich domain of TFHA interacts with both TBP and DNA and stabilizes TBP binding to the TATA element. The four-helix bundle domain of TFHA is positioned to interact with other proteins such as transcriptional activators. The crystal structure provides insight on the function and mechanism of TFHA. It was found that the B and y subunit of human TFHA can function in antirepression, whereas the a subunit is additionally required for activation (Ma et al., 1996). From the yeast TFHA structure and the sequence similarity between yeast and human TFHA subunits, it is possible that the B and 7 subunits of human TFHA fold into the B-sandwich structure that binds TBP and DNA, while the a subunit is required to form the four-helix bundle domain that binds transcriptional activators. TFIIB Human TFHB exists as a single polypeptide of 35 kDa (Ha et al., 1991). TFIIB has also been identified in Drosophila (Wampler and Kadonaga, 1992; Yamashita et al., 1992), yeast (Pinto et al., 1992), and archaebacteria (Ouzounis and Sander, 1992; Qureshi et al., 1995). TFHB enters the PIC after TBP binding to the TATA element and recruits RNA pol H and TFHF (Buratowski et al., 1989). Consistent with this role, TFIIB interacts directly with the TBP-DNA complex, pol H, and TFIIF (Buratowski et al., 1989; Maldonado et al., 1990; Ha et al., 1993; Fang and Burton, 1996). TFIIB is involved in start site selection in yeast (Pinto et al., 1992; Li et al., 1994). 20 repeats v hilt et the 113? W11 l Benton (linensi fists helices. insole linden) TFHB contains a zinc-ribbon motif at its amino-terminus and two imperfect direct repeats which form the carboxy-terminal core (Zhu et al., 1996; Barberis et al., 1993; Malik et al., 1993). The carboxy-terminal core of TFIIB (TFIIBc) is capable of binding the TBP-TATA complex, but is deficient in transcription, likely due to its inability to support efficient recruitment of the pol H/TFIIF complex (Barberis et al., 1993; Buratowski and Zhou, 1993; Ha et al., 1993; Yamashita et al., 1993). The three- dimensional structures of TFHBc and the TFIIBc-TBP-DNA complex have been solved (Bagby et al., 1995; Nikolov et al., 1995). Each repeat of TFch consists of five a- helices, which form a compact globular domain with structural similarity to cyclins involved in cell cycle control (Gibson et al., 1994; Jeffrey et al., 1995). TFHBc binds underneath and on one side of the TBP-DNA complex and it interacts with both TBP and DNA, as suggested by footprinting and photocrosslinking experiments (Lee and Hahn, 1995; Lagrange et al., 1996). The basic amino-terminal repeat of TFHB contacts the acidic carboxy-terminal “stirrup” of TBP. The bending of the TATA element by TBP allows TFHB to interact with the phosphodiester backbone of DNA both upstream and downstream of the TATA sequence. Therefore, similar to TFHA, TFIIB stabilizes the TBP-TATA interaction through both protein-protein contacts and protein-DNA contacts. Recent experiments demonstrate that TFHB is a sequence-specific DNA-binding protein that recognizes a novel sequence element found in certain promoters (Lagrange et al., 1998). TFIIB and TFHA bind to different sides of the TBP-DNA complex. Although missing from the crystal structure, the N-terminal zinc-ribbon domain is speculated to bind DNA in the vicinity of the transcription start site and may stabilize the melting of the promoter. 21 lflll RAH protei in ye; iseiat Drost 91 al., TFIIF Human TFHF is a heterotetrameric factor consisting of RAP30 (26 kDa) and RAP74 (58 kDa) subimits. RAP is an acronym for an RNA polymerase H-associating protein. The subunits of TFHF were originally identified on the basis of their affinity for immobilized pol H and shown to be essential for pol H transcription (Sopta et al., 1985; Burton et al., 1988). TFHF was later purified independently as an indispensable pol H initiation factor (Flores et al., 1988; Flores et al., 1989; Flores et al., 1990). TFIIF was also known in human cells as factor FC (Kitajima et al., 1990), in rat cells as factor By (Conaway and Conaway, 1991), in Drosophila cells as factor 5 (Price et al., 1989), and in yeast cells as factor g (Henry et al., 1992). cDNAs encoding TFIIF subunits have been isolated fiom human (Sopta et al., 1989; Finkelstein et al., 1992; A30 etal., 1992), Drosophila (Kephart et al., 1993; Frank et al., 1995; Gong et al., 1995), Xenopus (Gong et al., 1992), and yeast (Henry et al., 1994; Sun and Hampsey, 1995). TFHF in higher eukaryotes consists of two subunits, homologous to human RAP74 and RAP30 respectively. Yeast TFIIF has three subunits, the two larger ones (ng1 and ng2) are homologous to RAP74 and RAP30 respectively, while the third subunit (ng3) is also a component of yeast TFIH) and SWI/SNF complexes. Unlike IFGI and IFGZ, the TFG3 gene is not essential for yeast cell viability and ng3 protein is not required for transcription in vitro (Henry et al., 1992; Henry et al., 1994). ‘Two human leukemogenic proteins, AF -9 and ENL, share considerable sequence similarity with ng3 (Cairns et al., 1996). ng1 (also known as Ssu71) is implicated in the start site selection in yeast (Sun and Hampsey, 1995). 22 01 RNA POLYMERASE ll P 311 all P 311 PWJ. 311 NJ Rn Po Dammit] panorama parmmnusl panama-15H: m '3? \X l 41 P R RFC. R Pa 3 Po IO'CI use) m ECOLI RNA POLYMERASE Figure 1. A model for the wrapping of the promoter DNA around RNA polymerase during formation of the preinitiation complex. This model can account for the results obtained for both E. coli RNA polymerase and RNA polymerase II. The asterisks indicate isomerization of the complex. Abbreviations: P, promoter; R, E. coli RNA polymerase; Rn, RNA polymerase II; D, TBP; B, TFIIB; F30, RAP30; F30/74, RAP30/74; E, TFIIB; H, TFIHJ; RPCI, closed complex I; RPCZ, closed complex 11; RPO, open complex; RnPw‘ L, loosely wrapped complex; RnPw, T, tightly wrapped complex. 23 « I fit 11'... Primary sequence analysis indicates that RAP30 contains two distinct regions with sequence similarity to bacterial 0 factors (Sopta et al., 1989; Garrett et al., 1992). One of the o-homology regions in RAP30 is thought to interact with pol H, and TFIIF binds to the same surface on E. coli RNA polymerase as 0'70 does (McCracken and Greenblatt, 1991). Moreover, RAP30 has a crytic DNA-binding domain at its carboxyl terminus, a feature also shared by bacterial (:70 (I‘ an et al., 1994). The NMR structure of the C-terminal region of human RAP30 displays a helix-trun-helix fold, similar to the linker histone H5 and the hepatocyte nuclear transcription factor HNF-3/fork head (Groft etal., 1998). The N-terminal region of RAP30 binds RAP74 and TFHB (Fang and Burton, 1996). The RAP74 protein can be divided into three regions based on sequence analysis: the N-terminal globular domain, the C-terminal globular domain, and a flexible central region (Wang and Burton, 1995; Lei et al., 1998). No three-dimensional structure of RAP74 has been reported to date. The N-terminal domain of RAP74 binds RAP30 while the C-terminal domain binds TFIIB and pol H (Wang and Burton, 1995; Yonaha et al., 1993). The C-terminal domain of RAP74 also stimulates a CTD phosphatase activity that removes phosphate groups fi'om the CTD of the largest subunit of pol H (Chambers et al., 1995). This stimulation by RAP74 can be blocked by TFIIB, indicating a dynamic interaction between TFIIF, TFIIB, and CTD phosphatase. One important role of TFIIF is to deliver pol H to the preinitiation complex (Conaway et al., 1991; Flores et al., 1991; Killeen et al., 1992). Pol H alone cannot stably 24 pron RAP: hose _ 'th and 08h 3130 than Yin. he, associate with the TBP-TFHB-DNA complex (DB complex), and must be escorted to the promoter by TFIIF to form the TBP-TFHB-pol H-TFHF-DNA (DBPolF) complex. RAP30 alone in some cases is sufficient for delivering pol H to the DB complex, however, RAP74 is stimulatory and enhances the stability of the DBPolF complex (Killeen et al., 1992; Flores et al., 1991; Tyree et al., 1993; Tan et al., 1994). In another study, both RAP30 and RAP74 were found to be necessary for the formation of a DBPolF complex (Lei et al., 1998; Chapter 2). The regions of RAP30 and RAP74 that are critical for the interaction between these two polypeptides are both required for forming the DBPolF complex. TFHF plays a key role in transcriptional initiation after PIC assembly. Using photocrosslinking approaches, the locations of pol H and GTFs on the adenovirus major late promoter (AdMLP) were mapped (Coulombe et al., 1994; Forget et al., 1997; Robert et al., 1996; Robert et al., 1998). Surprisingly, TFHF induces a conformational change of pol H and wraps the promoter DNA around pol II and TFIIF (Forget et al., 1997; Robert et al., 1998; see Fig. 1). Both RAP30 and RAP74 are required for forming the wrapped complex. Analyses of RAP74 deletion mutants demonstrated that the activities of RAP74 fragments in wrapping the DNA correlate very well with their activities in transcriptional initiation and elongation (Lei et al., 1998; Robert et al., 1998). The DNA wrapping is also an important feature in prokaryotic transcription and is critical for the formation of a transcription-competent “open” complex (Schickor et al., 1990; Travers, 1990; Craig et al., 1995; Polyakov et al., 1995). In the pol H preinitiation complex, the DNA is first bent at the TATA box by TBP and likely bent again near the transcription start site due to the wrapping of promoter DNA around pol H. It is postulated that the 25 Figure 2. Proposed functions of TFIIF in transcriptional initiation and elongation. A) Domain structure of RAP74 in the TFIIF complex. The RAP74 N-terminal domain is drawn as three connected dark gray segments. Rows of X3 represent contacts between subunits. HHU and HRZ are proposed to include RAP74 dimerization domains. B) Preinitiation complex (PIC). TBP and the Coterminal repeats of TFIIB (IIB) are shown interacting with the TATA box. C) Isomerization of the PIC. D and E) Elongation complexes (EC). 26 Figure 2 27 RNA ; Prise e require little: BA 1 data d (bell Wit m3? thong. 512th. DNA theto [ltd bending and wrapping cause a superhelical torsion on the promoter and destabilize the two DNA strands near the start site. This strand unwinding then allows the access of TFHH helicase to separate the template strands in the presence of ATP to form an open complex. In addition to its role in initiation, TFHF also stimulates the elongation rate of RNA polymerase H (Bengal et al., 1991; Izban and Luse, 1992a; Kephart et al., 1994; Price et al., 1989; Tan et al., 1994; Lei et al., 1998). Both RAP30 and RAP74 are required for this stimulation (Lei et al., 1998; Tan et al., 1994). It was previously proposed that TF HF stimulates the elongation rate by suppressing transient pausing of the RNA polymerase (Bengal et al., 1991; Price et al., 1989; Izban and Luse, 1992a). Recent data demonstrate that TFIIF does not affect the position of pausing sites but decreases the dwell time of pol H at many sites (Chapter 3). Because the same region of RAP74 is required for the activities of TFHF in DNA wrapping, initiation, and elongation, it is suggested that the DNA template is also wrapped around pol H and TFHF during elongation to maintain the open complex and therefore enhance the rate of RNA synthesis (Lei et al., 1998; Robert et al., 1998; Fig. 2). In the elongation complex, the DNA template may either be fully wrapped (Fig. 2D) or partially wrapped (Fig. 2B) but the torsional strain at the position of phosphodiester bond formation is maintained in both cases. . TFIIF is also involved in the recycling of pol H (Lei et al., 1998). TFIIF stimulates a CTD phosphatase activity which converts pol 110, the elongation form of pol II, to pol HA, the initiation form (Chambers et al., 1995). The C-terminal domain of 28 RAP din 311E llil RAP74 is suflicient for this stimulation. It was recently demonstrated that the C-terminal domain and the central region of RAP74 are necessary for continuous RNA initiation in an extract system, possibly by stimulating the CTD phosphatase activity to regenerate pol HA (Lei et al., 1998; Chapter 4). RAP7 4 is highly phosphorylated in vivo, consistent with the presence of many putative phosphorylation sites in its central region (Sopta et al., 1985; Burton et al., 1988; Flores et al., 1989; Finkelstein et al., 1992). Many kinases including TAFn250 (Dikstein et al., 1996) and TFIIH (Malik et al., 1998) can phosphorylate RAP74 in vitro. Both the initiation and elongation activities of TFHF are moderately stimulated by the phosphorylation of RAP74 (Kitajima et al., 1994). However, the phosphorylation of RAP74 is not required for either initiation or elongation because recombinant RAP30 and RAP74 produced in E. coli are active in a highly purified system which do not contain either TFIH-I or T AF us (Malik et al., 1998; Chapter 5). TFIIE TFHB enters the PIC after pol H and HF and recruits TFIIH in vitro (Buratowski et al., 1989; Flores et al., 1989; Flores et al., 1992; Maxon et al., 1994). Consistent with this role, TFHB interacts directly with pol H, TFIIF, and TFIIH (Flores et al., 1989; Maxon and Tjian, 1994). The cDNAs encoding TFHB proteins have been cloned from human (Ohkuma et al., 1991; Peterson et al., 1991; Sumimoto et al., 1991) and yeast (F eaver et al., 1994). Similar to TFHF, Human TFIIE is a heterotetramer of 56 kDa (HEa) and 34kDa (HEB) subunits (Ohkuma et al., 1990; Inostroza et al., 1991). Yeast TFHB may exist as an heterodimer (Leuther et al., 1996). TFHEa has a zinc-ribbon motif 29 C01 0011 and yeast TFHB binds single-stranded DNA, which suggests that TFHB may participate to form or stabilize the melted DNA in the initiator region (Kuldell and Buratowski, 1997). The fimctions of TFHE include recruitment of TFIHI to the PIC, stimulation of the CTD kinase activity of TFHH, and stimulation of the ATP—dependent DNA helicase activity of TFHH (Lu et al., 1992; Ohkuma and Roeder, 1994; Ohkuma et al., 1995). Additionally, TFHB contributes to DNA wrapping within the PIC (Robert et al., 1996; Robert et al., 1998). Two-dimensional crystallography of a TFHB-pol H complex suggests that TFIIE promotes a conformational switch at the active center upon pol H- DNA interaction (Leather et al., 1996). TFHB has also been implicated as the direct target of some transcriptional regulators (Sauer et al., 1995; Zhu and Kuziora, 1996). TFHH The entry of TFIH-l completes the assembly of a preinitiation complex (Flores et al., 1992). TFIH-I was identified fiom human, rat, and yeast cells (Conaway and Conaway, 1989; Flores et al., 1992; Gerard et al., 1991). TFIHI is a multisubunit complex and contains several enzymatic activities including a CTD kinase and two ATP- dependent DNA helicases (reviewed in Drapkin and Reinberg, 1994; Orphanides et al., 1996). TFIH-I is required for transcriptional initiation fiom linear DNA in vitro. TFIH-l has a critical role in forming the open transcription complex (Dvir et al., 1996; Holstege et al., 1996). One of the two DNA helicases in TFIH-I, namely ERCC3 in human and Rad25 in yeast, is essential for transcriptional initiation and believed to be necessary for promoter melting to form the “open” complex (Park et al., 1992; Feaver et al., 1993; 30 / Guzder et al., 1994a; Guzder et al., 1994b; van Vuuren et al., 1994). The requirement for TFIIE, TFHH, and ATP can be bypassed when transcribing fiom the DNA template either with a high negative superhelical density, which favors DNA unwinding, or with an artificially premelted region at the start site (Parvin and Sharp, 1993; Tyree et al., 1993; Holstege et al., 1995; Pan and Greenblatt, 1994). These experiments directly link TFHB, TFIH-I, and ATP to promoter melting. TFIH-I is also important for the transition form initiation to elongation, likely mediated by the CTD kinase activity of TFHH. Presumably, phosphorylation of the CTD causes a conformational change in the PIC, disrupting the interaction between the CTD and TBP and leading to promoter clearance (U sheva et al., 1992; Goodrich and Tjian, 1994). TFHH also promotes the transition from very early elongation complexes to stable elongation complexes (Dvir et al., 1997). The downstream DNA sequence to position +40 is required for promoter escape, perhaps because DNA is wrapped around TFIIH during this transition. In addition to its roles in transcription, TFIHI is also implicated in nucleotide excision repair (NER) and cell cycle progression (Drapkin et al., 1994a; Drapkin et al., 1994b; Orphanides et al., 1996). First, five of the nine subunits of TFIH-I for which cDNAs have been isolated have dual roles in transcription and NER. It is proposed that TFHH plays a central role in coupling transcription and DNA repair (Drapkin et al., 1994c; Friedberg, 1996; Sancar, 1996). Second, human TI-IHH also contain a CAK (cdk- activating kinase) complex consisting of the protein kinase cdk7 (also known as .MOlS), its cyclin partner cyclin H, and an activating protein MAT-l (reviewed in Orphanides et al., 1996). The CAK complex is thought to play a pivotal role in cell-cycle regulation by 31 activating the cyclin-dependent kinases (cdks). The cdk7 kinase is required for transcription fiom the TATA-less DI-IFR promoter, perhaps at the stage of promoter clearance (Akoulitchev et al., 1995). However, a direct role for TFIH-I in cell-cycle progression remains controversial. TFIHI kinase activity does not vary during the cell cycle, perhaps due to the association of MAT-l with cdk7 and cyclin H which makes cdk7 constitutively active (Adamczewski et al., 1996). Moreover, the CAK complex is not tightly associated with other TFIH-I subunits and in fact only 20% of the total cellular CAK activity is associated with TFIIH (Drapkin et al., 1996). Furthermore, although the yeast TFIH-I kinase Kin28 phosphorylates the CTD and is required for transcription, it does not have CAK activity in vitro (Feaver et al., 1994; Cisrnowski et al., 1995). Rather, the yeast CAK activity resides in a single polypeptide which does not phosphorylate the CTD and is not a component of TFIIH (Kaldis et al., 1996; Kaldis et al., 1998). The yeast CAK and human CAK appear to have different substrate specificities (Kaldis et al., 1998). TRANSCRIPTIONAL INITIATION Transcriptional initiation by pol H proceeds through four stages: PIC assembly, open complex formation, abortive initiation, and promoter escape or promoter clearance (Zawel and Reinberg, 1995). Unlike E. coli RNA polymerase, eukaryotic pol I or pol HI, the initiation of transcription by pol H is dependent on energy, requiring hydrolysis of the B-y phosphoanhydride bond of ATP (Bunick et al., 1982; Lofquist et al., 1993). The ATP hydrolysis presumably supports the helicase activity of TFIH-I to form the open complex (Holstege et al., 1996). 32 Pll 111C fro TA PIC Assembly The assembly of PIC is a prerequisite for transcriptional initiation to occur. Two models, the step-wise assembly model and the holoenzyme model, have been proposed. Previous in vitro experiments have led to a step-wise model for the assembly of a PIC: l) recognition of the TAT A element by TBP; 2) binding of the TBP-promoter complex by TFHB; 3) recruitment of pol H and TFIIF; 4) recruitment of TFHB; and 5) recruitment of TFHH by TFHE to complete the PIC formation (Zawel and Reinberg, 1993). Although TFHA is not required for forming a PIC, TF HA can enter the complex after the binding of TBP and incorporation of TFHA stabilizes the complex. This sequential assembly model implies that transcriptional regulators can affect the assembly process by affecting any single step or multiple steps through interacting with one or more general factors (Choy and Green, 1993). Indeed, in vitro interactions between activators and TBP, TFHA, TFHB, TFIIF, TFHB, and TFIH-I have all been reported (reviewed in Triezenberg, 1995). On TATA-containing promoters, TBP is sufficient to nucleate the formation of a transcription-competent PIC and direct initiation. However, TBP is not sufficient for initiation fiom TATA-less promoters. TFHD instead of TBP is required for initiation from TATA-less promoters (Weis and Reinberg, 1992; Martinez et al., 1998). Other polypeptides within the TFHD complex (TAFns) may recognize the Inr and DPE of TATA-less promoters to initiate the preinitiation complex assembly. However, as mentioned earlier, it is unclear whether TAFns can coexist with pol H and other GTFs in 33 the 1113 Ind; D.\ EVE the transcription complex because TAFns appear to occupy the same DNA sequence as pol H and the GTFs do. The step-wise model for assembly of a PIC has been challenged recently by the discovery that a subset of the GTFs exist in a preassembled form in an RNA pol H “holoenzyme”, which suggests that most of the initiation machinery can bind to a promoter in a single step (Koleske and Young, 1995). The pol H holoenzymes contain transcriptional coactivators and respond to transcriptional activators when supplemented with TBP and the remaining GTFs. One important implication is that a substantial portion of transcriptional control is mediated directly through the basal transcriptional mechanism and does not require TAFns. Although the components of the SRB/mediator- containing pol H holoenzyme are well defined both genetically and biochemically, the mammalian holoenzymes are not yet well characterized. Different preparations of mammalian pol H holoenzymes appear to contain very different components, such as DNA repair proteins, splicing and polyadenylation factors, histone acetyltransferases, or even the breast cancer tumor supressor BRCAl (Maldonado et al., 1996b; McCracken et al., 1997; Scully et al., 1997 ; Cho et al., 1998). Therefore, two questions need to be addressed: 1) does each mammalian holoenzyme complex reported so far represent a homogenous complex or many hetero genous subcomplexes? and 2) what is the relative abundance of the holoenzyme compared with the fi'ee pol H and GTFs inside the cells? Since numerous GTF-GTF interactions and GTP-pol 11 interactions have been reported, it should not come as a surprise that GTFs and pol H can associate when not bound to promoter DNA (Conaway and Conaway, 1993). One concern of the various affinity chromatography approaches used for isolating pol H holoenzymes is that the immobilized 34 ligand may serve as a surface to assemble holoenzyme complexes fi'om relatively fi'ee cdmponents during the process of isolation. More extensive and careful biochemical analyses have to be carried out to firmly establish the presence of distinct mammalian pol H holoenzymes in vivo. My view is that the assembly of pol H holoenzyme complexes inside cells is dynamic and transient, and the assembly process itself may be subject to regulation by transcription factors and signal transduction pathways. Interestingly, under some circumstances, RNA pol H can accurately initiate transcription in the absence of a full complement of general transcription factors. For instance, initiation can occur in the absence of ATP, TFHB and TFIH-I on supercoiled templates (Parvin and Sharp, 1993; Tyree et al., 1993). Using a superhelical IgH promoter, TFHF is also dispensable (Parvin and Sharp, 1993). Because negative supercoils facilitate the unwinding of DNA strands, these observations are interpreted as evidences for the involvement of TFIH-I, TFIIE, and ATP in promoter melting to form an open complex on linear DNA template. The roles of TFIIE, TFIH-I, and ATP hydrolysis in promoter melting were further supported by elegant experiments using an artificially “premelted” promoter as the template. When using a premelted promoter with a short region of mismatched heteroduplex DNA at the start site, the transcription becomes independent of TFHE, TFIH-I, and hydrolyzable ATP (Holstege et al., 1995; Pan and Greenblatt, 1994). These results suggest that the ATP-dependent DNA helicase activity of TFIH~I is essential for promoter melting when using linear DNA templates. Besides recruiting TFHH, TFHB may play a direct role in promoter melting in the absence of TFIH-I (Holstege et al., 1995). 35 Another minimal transcription system has also been reported. Although it is generally believed that TBP is required for transcription from all eukaryotic promoters in vivo, transcription from the adeno-associated virus (AAV) P5 promoter in vitro can occur independent of TBP (U sheva and Shenk, 1994). In this case, YYl (an Int-binding protein), TFHB, and pol H are sufficient to direct basal transcription fi'om a supercoiled template. YYl binds within the AAV-P5 Inr and cooperates with TFHB to recruit pol II to the promoter. TFHB was recently identified as a sequence-specific DNA binding protein (Lagrange et al., 1998). It will be interesting to test whether the DNA-binding activity of TFHB is required for transcription in this minimal system. Although TBP is usually associated with additional polypeptides, such as TAFns in the TFHD complex, a TBP-free-TAF-containing complex (TFTC) was recently isolated fi'om human cells (W ieczorek et al., 1998). This complex contains many known TAFns and some unidentified polypeptides but does not have TBP. The most intriguing feature of the TFTC complex is that, when supplemented with pol H, TFHB, TFHE, TFHF, and TFIH-I, it can direct specific initiation from several promoters, both TATA- containing and TATA-less. The addition of recombinant TBP only stimulated the level of transcription. Because a TBP-related factor (TRF) has been reported (Hansen et al., 1997), it is important to identify the tmknown polypeptides in the TFTC complex and determine if a TBP-like function resides on one of these unidentified polypeptides. The isolation of TFTC and the presence of TAFus in other protein complexes, such as the mammalian P/CAF complex (Ogryzko et al., 1998) and the yeast SAGA complex (Grant et al., 1998), reemphasize the concept of dynamic interaction among transcription factors. The specific functions of each TAFu in these complexes need to be further determined. 36 Open Complex Formation In order to gain access to the nucleotides of the template strand, DNA-dependent RNA polymerases require separation of the two DNA strands prior to initiation of transcription. The DNA strand separation can be detected by modifications of single- stranded DNA with chemical reagents such as KMnOa that reacts with single-stranded thymidines (Holstege et al., 1996; Holstege et al., 1997; Jiang et al., 1995; Jiang et al., 1996). A prerequisite for pol H promoter opening is the presence of ATP hydrolyzable at the B-7 bond (Wang et al., 1992; Jiang et al., 1993). This is a unique feature of pol H transcription because other DNA-dependent RNA polymerases, including eukaryotic pol I and pol 1H, and bacterial RNA polymerases, do not require ATP hydrolysis for initiation (Bunick et al., 1982; Lofquist et al., 1993; Eick et al., 1994). Promoter opening at the adenovirus major late promoter requires formation of the complete PIC containing TBP, TFHB, TFHF, TFHB, TFIHI, and pol H (Holstege et al., 1996). The promoter opening occms in two steps: first, dependent on ATP but prior to initiation, the --9 to +1 region becomes single-stranded; and second, formation of the first phosphodiester bond results in expansion of the open region to the +8 position. These observations lead to the model that the TFHH-associated DNA helicase activity is crucial for creating a single-stranded region during formation of the open complex. This then gives pol H access to the nucleotides of the template strand and allows expansion of the open region upon formation of the first phosphodiester bond (Holstege et al., 1996). By analyzing the open regions and RNA products of various stalled elongation complexes, it was further shown 37 that the TFIH-I helicase activity is required for maintaining the open region before formation of a four-nucleotide RNA product (Holstege et al., 1997). . TFIH-I is the only general transcription factor known to possess ATP-dependent enzymatic activities: a CTD kinase and two DNA helicases (ERCC-2 and ERCC-3) (Feaver et al., 1991; Feaver et al., 1993; Lu et al., 1992; Serizawa et al., 1992; Schaeffer et al., 1993; Schaeffer et al., 1994; reviewed in Drapkin and Reinberg, 1994b). However, the CTD kinase activity of TFHH is not generally required for initiation fi'om all promoters tested. For example, the CTD kinase activity of TFIIH plays no role in transcription fiorn the TATA-containing AdMLP although it is required for initiation from the TATA-less DI-IFR promoter (Serizawa et al., 1993; Makela et al., 1995; Akoulitchev et al., 1995). Therefore, the general requirement for B-y bond hydrolysis of ATP may result fi'om the TFIH-I-associated DNA helicase activities for promoter opening. It was recently shown that one of the TFHH-associated DNA helicases, ERCC- 3, is essential for transcription while the ERCC-2 helicase is required for DNA repair (Drapkin and Reinberg, 1994b; Orphanides et al., 1996). Using negative supercoiled templates and premelted templates allowed the requirement for TFIIE, TFIHI and ATP to be bypassed, which further supported the conclusion that TFIHI, TFIIE, and ATP function in promoter opening. TFIIE is implicated in promoter opening because it recruits TFIHI into the PIC (Flores et al., 1992). In addition to its function as a tethering factor, TFHB stimulates transcription fiom negatively supercoiled templates independent of TFIH-I (Tyree et al., 1993). This stimulatory effect of TFIIE is increased by conditions that enhance the stability of the DNA duplex and therefore make the DNA more resistant to opening, thus 38 implicating TF HE in promoter melting in a direct manner (Holstege et al., 1995). TFIIE binds single-stranded DNA, possibly through the zinc-ribbon motif of TFHE-ct, the large subrmit of TFIIE (Kuldell and Buratowski, 1997). TFHE may participate to form or stabilize the single-stranded DNA in the melted region. Recent photocrosslinking studies indicate that TFHE stabilizes the wrapping of promoter DNA around pol H and TFHF in the PIC (Robert et al., 1998; Robert et al., 1996). It is proposed that the bending and wrapping of DNA have a direct consequence for topological untwisting of the helix around the transcription start site (Robert et al., 1998). Because TFHF is essential for forming the wrapped structure in the PIC, these studies implicate TFHF in formation of the open complex. Abortive Initiation Similar to bacterial RNA polymerases, RNA polymerase H goes through an abortive phase of transcription in which short RNA transcripts are synthesized and released before productive elongation occms (Luse and Jacob, 1987). The abortive initiation may reflect a conformational change that has to occur to convert the initiation complex to a productive elongation complex. It may serve as a checkpoint for pol H and transcriptional regulators before pol H embarks on elongating a full length RNA transcript. Promoter Escape or Promoter Clearance Promoter escape, also called promoter clearance, is the transition from abortive initiation to productive elongation. Many promoters, such as Drosophila hsp70 and 39 mammalian c-myc, appear to be regulated at the step of promoter escape in vivo (Lee et al., 1992; O. Brien et al., 1994; Krumm et al., 1995). It is believed that pol H must break some protein-protein contacts and protein-DNA contacts that hold it on the promoter to commence elongation. Presumably, this transition requires a conformational change in pol H and release of some initiation factors. For instance, promoter escape in bacterial systems involves the release of a factor and significant changes in polymerase comformation and template contacts (Metzger et al., 1989). In the pol H system, it is suggested that the phosphorylation of the pol H CTD contributes to promoter escape. As mentioned earlier, pol H enters the preinitiation complex with an unphosphorylated CI'D and the pol H CI'D is highly phosphorylated in the elongation complex. Moreover, the phosphorylation of the CTD occurs concomitant or shortly after initiation. The phosphorylation of the CTD may break some protein contacts because the mphosphorylated CI‘D interacts with several general transcription factors including TBP and TFHE (Flores et al., 1989; Usheva et al., 1992). It is not clear which general transcription factors are present in the elongation complex after initiation and promoter escape. Using a template competition approach in a reconstituted system containing pol H, TBP, TFHB, TFHF, TFHE, and TFIH-I, it was shown that after initiation, all general transcription factors dissociate from pol H (Zawel et al., 1995). Only TFHF can reassociate with the elongation complex when pol H encounters pausing sites and suppresses pausing. This implies that TFIIF is the only general factor that functions in both initiation and elongation, and other general factors function only as initiation factors. However, it is unclear which general transcription factors accompany pol H in the elongation complex in vivo. For example, other factors 40 that normally hold some of the general factors in the elongation complex may be missing in this reconstituted system. It was recently found that recombinant TBP, TFHA, TFHB, and TFHE can stimulate the elongation rate of pol H in cooperation with TFHF (Chapter 5). These factors may recruit and stabilize TFIIF in the elongation complex through protein-protein contacts and possibly protein-DNA contacts. It is possible that, like in the PIC, these factors function in concert with TFHF to wrap the DNA around pol H to maintain the transcription bubble during productive elongation (Robert et al., 1998). It remains important to test whether the elongation stimulation by TBP is TATA element- dependent or it can happen when the elongation complex is away hour the promoter. It is also important to find out whether any of these general factors does exist in the elongation complex in vivo. TRANSCRIPTIONAL ELONGATION For many genes transcribed by pol H, elongation is rate limiting for the production of full-length transcripts (reviewed in Bentley, 1995). Transcriptional control at the level of elongation has been observed in many viral transcriptional units such as the HIV-1 and cellular genes such as c-myc, c-myb, c-fos, and hsp70. Mechanics of Elongation Two models have been proposed for explaining the mechanism of transcript elongation: monotonic versus “inchworming” (reviewed in von Hippel, 1998). It was proposed that the elongation complex moves mostly in a monotonic fashion and enters 41 the “inchworming” cycle only when it encounters specific signals in the DNA template and the nascent RNA (Nudler et al., 1994). A central feature of the elongation complex is a transiently open transcription bubble (~ 18 bp in length) which moves with the RNA polymerase through the otherwise double-stranded DNA while the polymerase catalyzes template-directed transcript elongation (von Hippel, 1998). Elongation of transcription has been perceived as a monotonous process in which each nucleotide addition is accompanied by a one-base pair translocation of RNA polymerase, which was considered as an inflexible and rigid structure. However, recent observations suggest a substantial conformational flexibility of the ternary elongation complex. The DNA:RNA hybrid, protein-DNA contacts, and protein-RNA contacts all contribute to the stability and movement of the elongation complex (Nudler et al., 1996; Nudler et al., 1997; Nudler et al., 1995). E. coli RNA polymerase is proposed to have two RNA-binding sites: a 3’-proximal “loose” RNA- binding site, and an upstream “tight” RNA-binding site. A third flexible element within the polymerase, termed the “fi‘ont-end domain”, contacts the double-stranded DNA just downstream of the transcription bubble (Nudler et al., 1994). The elongation complex tends to stay in a preferred, relaxed conformation as it advances along the template. As the RNA chain grows, the filling of the loose binding site alternates with the threading of the recently synthesized RNA through the tight binding site so that an optimal distance between the catalytic center and the fiont end is maintained. However, certain sequence signals encountered on the way cause the ternary complex to rearrange. The rearrangement is induced by the “anchoring” of the fi'ont-end domain to the DNA and accompanied by the cessation of threading of the newly synthesized RNA through the 42 tight RNA-binding site. At the same time, the growth of the RNA chain continues, resulting in progressive filling of the loose RNA-binding site. This causes a buildup of internal strain within the elongation complex. The strained conformation is reverted to the relaxed form only when the anchoring contacts are broken and the fi'ont end leaps forward with simultaneous threading of the transcript through both RNA-binding sites, a process termed “inchworming”. By comparing stalled complexes at many positions, it is shown that the inchworming cycle is an incidental event rather than intrinsic to elongation. In the absence of the signal, the RNA polymerase translocates concomitantly with the growth of the transcript and the elongation proceeds in a monotonous fashion. Only when encountering the signal, the ternary complex becomes strained and the inchworming cycle occurs. Entering an inchworming cycle may be a mechanism to slow elongation for entry into pause, arrest, or termination mode. Although the discontinuous mechanism of transcriptional elongation is mainly derived from the study of bacterial RNA polymerases, a similar mechanism also appears to be utilized in eukaryotic systems. Conformational changes in pol H accompanying “inchworm” movement have been demonstrated by probing isolated ternary complexes with nucleases and RNA cleavage factors (Izban and Luse, 1993a; Linn and Luse, 1991). E. coli RNA polymerase apparently switches fiom monotonic progress to the inchworm mode in response to DNA sequences that lock the loose RNA-binding site and the front- end domain of polymerase onto the DNA (Nudler et al., 19945. It is likely that pol H behaves in a similar way druing elongation. 43 Pausing, Arrest, and Termination Under physiological conditions, the optimal elongation rate of RNA polymerases is about 1,200-1,500 nucleotides (nt)/min in higher eukaryotes (U cker and Yamamoto, 1984; Izban and Luse, 1992a). Furthermore, many of the genes transcribed are up to 104 or 105 bp in length, demanding a high processivity of the elongation complex. During elongation, however, intrinsic signals in template DNA and nascent RNA can divert a fraction of RNA polymerases fiom the path of rapid and processive elongation (reviewed in Landick, 1997). Three types of such signals are: I) pause signals, which can stop the RNA polymerase temporarily to await interaction of a regulatory molecule, but from which the elongation complex can escape spontaneously; 2) terminators, which can irreversibly release RNA and DNA fi‘om the RNA polymerase (RNA polymerases never I extend released transcripts; to the contrary, DNA polymerases can rebind and extend the 3’ ends of released DNA molecules); and 3) arrest signals, which can cause RNA polymerase to backtrack its catalytic center to an internal phosphodiester in the nascent RNA chain. The escape fiom an arrest site then requires hydrolysis of the transcript, catalyzed by the RNA polymerase, to realign the 3’ end of the nascent RNA and the catalytic center correctly. This RNA cleavage reaction is stimulated by GreA or GreB in bacteria and by SH in eukaryotes (Borukhov et al., 1993; Borukhov et al., 1992; Izban and Luse, 1993a; Izban and Luse, 1992b). During elongation, therefore, RNA polymerase must hold the DNA template and nascentRNA tightly enough to avoid dissociation yet loosely enough to translocate rapidly along the template, and it must be able to reverse these properties efiiciently at the template positions where pausing or termination is programmed. Although multiple classes of pause, termination, and arrest signals are identified in bacterial systems, the signals in eukaryotes are poorly understood (Landick, 1997; von Hippel, 1998). For all RNA polymerases at each position in a gene, the ternary complex may have a slightly different structure induced by the template sequence that affects the energetic barriers to the polymerization reaction. As a result, enormous variation is seen in the polymerase “dwell time” at different positions (von Hippel and Yager, 1992). The difl‘erence in dwell time may reflect the difference in KIn for NTP at the next position. Pauses can be affected by many factors including the DNA sequence, DNA bending, nucleotide availability and misincorporation, RNA secondary structure, and DNA- binding proteins that may act as roadblocks. Pausing appears to be a universal prerequisite for termination, but certainly not all pause sites are termination sites. Although the mechanism is still unclear, the discontinuous movements of RNA polymerases may mediate steps in pausing, termination, and arrest (Nudler et al., 1994; Landick, 1997). Both the DNA:RNA hybrid and the protein-nucleic acid contacts within the ternary complex are key determinants in these processes. Elongation Factors Several general pol H elongation factors have been identified from human, yeast, rat, and Drosophila (Reines et al., 1996). These include TFIIF, SH, SHI, ELL, P-TEFb, and N-TEF. Among these factors, TFIIF, SHI, and ELL stimulate the elongation rate and suppress transient pausing of pol H. SH stimulates the intrinsic RNA cleavage activity of 45 pol H to overcome pause sites and arrest sites. P-TEFb (positive elongation factor b) increases the processivity of pol H, while N-TEF (negative elongation factor) causes premature termination. One component of N-TEF, namely Drosophila factor 2, is an ATP-dependent transcript release factor and a member of the SWIZ/SNFZ ATPase family (Xie and Price, 1997; Liu et al., 1998). The human homolog of factor 2 also displays the double-stranded DNA-dependent ATPase activity and induces pol H termination (Liu et al., 1998). TFHF has dual roles in both initiation and elongation (Bengal et al., 1991; Izban and Luse, 1992a; Kephart et al., 1994; Tan et al., 1994; Lei et al., 1998). Both RAP30 and RAP74 subunits are required for supporting initiation and stimulating the elongation rate (Tan et al., 1994; Lei et al., 1998). It was suggested that TFHF stimulates the elongation rate by suppressing the transient pausing of pol H (Bengal et al., 1991; Izban and Luse, 1992a; Price et al., 1989). TFHF does not appear to afi‘ect the positions of pausing but generally decreases the dwell time of pol H at each position (Chapter 3). By analyzing a series of TFHF mutant proteins in elongation, it is clear that the activity of TFHF in stimulating elongation rate correlates very well with its ability in decreasing the dwell time of pol H at each pause site. However, the cause-effect relationship between the rate stimulation and the pause suppression of TFHF activities is still unclear. Biochemical and genetic evidences suggest that SH (also known as TFHS or was) interacts with pol H (Sopta et al., 1985; Agarwal et al., 1991; Archambault et al., 1992). SH does not enhance the elongation rate but promotes the passaging of pol H through intrinsic DNA arrest sites and pause sites (Bengal et al., 1991; Izban and Luse, 1992a; Reines et al., 1989; Reinberg and Roeder, 1987). SH stimulates an endonuclease 46 activity of pol H that hydrolyzes the nascent RNA, resetting the pol H catalytic center at the 3’ end of the transcript and allowing elongation to resume (Izban and Luse, 1992b; Johnson and Chamberlin, 1994; Powell et al., 1996). The increment of SH-facilitated transcript cleavage varies dramatically between paused and arrested ternary complexes, indicating that paused complexes and arrested complexes have distinct conformations (Izban and Luse, 1993a; Izban and Luse, 1993b). The pol H subunit pr9 appears to interact with SH to regulate the transcript cleavage and the read-through activities of pol H (Awrey et al., 1997). By stimulating the intrinsic 3’-5’ RNA cleavage activity of pol H, SH also enhances the fidelity of transcription (J con and Agarwal, 1996; Yoon et al., 1998). P-TEFb was originally identified fi'om Drosophila as a factor important for the production of DRB-sensitive transcripts in vitro (Marshall and Price, 1995). DRB, an ATP analog and kinase inhibitor, is known to inhibit elongation both in vitro and in vivo (Zandomeni et al., 1983; Zandomeni and Weinmann, 1984; Chodosh et al., 1989; Yankulov et al., 1995). The human homolog of P-T'EFb was also identified (Peng et al., 1998a). Both Drosaphila and human P-TEFb are cyclin-dependent kinases which can specifically phosphorylate the CTD of pol H (Marshall et al., 1996; Peng et al., 1998a; Peng et al., 1998b). In addition to its role as a general elongation factor, P-TEFb also has a specific fimction in the HIV -1 Tat-mediated transcriptional activation (Zhu et al., 1997; Fujinaga et al., 1998). Tat, a virally encoded transcriptional activator, is required for the synthesis of full-length RNA molecules from the HIV-1 virus genome (reviewed in Jones and Peterlin, 1994). In the absence of Tat, short transcripts are formed and released, a process called premature termination. HIV-1 Tat is a unique transcriptional activator 47 because, instead of bindng a DNA element, it binds an RNA sequence (termed TAR) which is located near the 5’ end of the nascent transcript. The large subunit of P-TEFb, cyclin T, interacts specifically with HIV-1 Tat and enhances the affinity and specificity of the Tat:TAR interaction (Wei et al., 1998). The interaction between Tat and P-TEFb in tmn also recruits P-TEFb to the paused early elongation complex. Presumably, the CTD kinase activity of P-TEFb can then phosphorylate the pol H CTD and convert the paused elongation complex to a processive elongation complex. Alternatively, the kinase activity of P-TEFb can phosphorylate another transcription factor that is specifically required for productive transcription of the HIV-1 genome. SH] and ELL have similar functions to TFIIF in stimulating the overall elongation rate and suppressing the transient pausing of pol II (Bradsher et al., 1993; Bradsher et al., 1993; Shilatifard et al., 1996; Shilatifard et al., 1997a; Shilatifard et al., 1997b). Interestingly, both ELL and SIH are implicated in the development of certain human cancers. The gene encoding ELL is a fi'equent target for t(11; l9) chromosomal translocations in acute myeloid leukemias (Shilatifard et al., 1996). SHI, also called elongin, is a target for regulation by the protein product of the VHL (von Hippel-Lindau) tumor suppressor gene, which is mutated in several types of tumors (Aso et al., 1995; Duan et al., 1995). The normal tumor suppressor function of the VHL protein likely involves the down-regulation of SHI (elongin) transcriptional activity. SHI (elongin) consists of a transcriptionally active A subunit and two regulatory B and C subunits. The elongin B and elongin C subunits form a stable binary complex that interacts with elongin A and strongly induces its elongation activity (Takagi et al., 1996; Aso et al., 1996). The VIH. tumor suppressor protein is capable of binding stably to the elongin BC complex 48 and preventing it from activating elongin A (Pause et al., 1996; Takagi et al., 1997; Conaway et al., 1998). The interaction of the VI-H. protein with the BC complex is mediated in part by a short VHL region and many naturally occming VHL mutants found in tumors have mutations that fall within this critical region. A number of these VHL mutants exhibit reduced binding to elongin BC complex in vitro. TRANSCRIPTIONAL TERMINATION AND REMIATION The signal and mechanism of termination in pol H transcription are poorly understood. It is possible that the dephosphorylation of the CTD may serve as a trigger for pol H to terminate, although this remains to be further tested. Drosophila and human ATP-dependent transcript release factors have recently identified. Both factors exhibit dsDNA-dependent ATPase activities and induce pol H termination from stalled elongation complexes (Xie and Price, 1996; Xie and Price, 1997; Liu et al., 1998). The CTD of pol H in the elongation complex is highly phosphorylated (Dahmus, 1996), however, pol HA is preferentially associated with the preinitiation complex (Lu et al., 1991). Therefore, the CTD has to be dephosphorylated so that pol H can re-enter the preinitiation complex for a successive round of transcription to occur, a process called transcriptional recycling. It should be noted that the transcriptional recycling is only one form of transcriptional reinitiation. Transcriptional reinitiation in vitro can have different forms: 1) the same DNA template is “fired” multiple times. As soon as the first polymerase molecule exits the promoter, a second preinitiation complex then assembles on the same template and new initiation occurs; 2) the same pol H molecule “fires” multiple times, i.e., transcriptional recycling. After finishing the synthesis of one RNA 49 chain, the same polymerase molecule is re-delivered to a promoter and initiates another round of transcription; and 3) difi‘erent pol H molecules ‘ e” from different DNA templates at difl‘erent times. Although they are mechanistically distinct, one shared requirement by these different forms of reinitiation is believed to be the conversion of pol HO to pol HA. In the case of transcriptional recycling, the dephosphorylation occms on a transcriptionally active polymerase right before or shortly after termination. In the two other cases, the CTD dephosphorylation may occur on fine polymerases to maintain the pool of pol HA. Indeed, the CTD phosphatase is capable of dephosphorylating the pol H CTD in the absence of DNA (Chambers and Dahmus, 1994). The yeast SrblO CI‘D kinase inhibits transcription by phosphorylating the pol H CTD prior to formation of the initiation complex on promoter DNA and therefore preventing PIC assembly (Hengartner et al., 1998). Cdk8, the mammalian homolog of SrblO kinase, is shown to be associated with an mammalian pol H holoenzyme and may also phosphorylate the CTD independent of DNA (Cho et al., 1998). Mammalian TFHH can also phosphorylate the pol H CTD independent of DNA although the yeast TFHH kinase Kin28 preferentially phosphorylate the CTD only when the transcription apparatus is associated with DNA (Hengartner et al., 1998). Therefore, it is likely that the phosphorylation status of pol H is tightly controlled by both CTD kinases and CI'D phosphatase to regulate the ablmdance of pol HA. Interestingly, the CTD phosphatase is stimulated by the RAP74 subunit of TFHF and TFIIB blocks this stimulation (Chambers et al., 1995). The C-terminal domain of RAP74 is sutiieient for stimulating CTD phosphatase activity. Consistent with its ability to stimulate the CTD dephosphorylation, firll length RAP74 supports multiple rounds of transcription in an extract system. RAP74 mutants containing only the N-terminal 50 domain do not stimulate the CTD dephosphorylation and are incapable of transcriptional reinitiation (Lei et al., 1998; Chapter 4). Because the CTD phosphatase, TFHF, and TFHB all bind pol H directly and function in the dephosphorylation reaction, it is possible that these factors cooperate to regulate transcriptional reinitiation by controlling the conversion of pol HO to pol HA. Additional factors may also be involved. The inhibitory effect of TFHB on CTD phosphatase activity may serve as a brake during elongation to prevent premature dephosphorylation, which would be expected to cause termination. TRANSCRIPTIONAL ACTIVATION In eukaryotic cells, packaging of DNA into chromatin causes overall gene repression (Paranjape et al., 1994). DNA sequences within the chromatin structure are generally inaccessible to transcription factors and pol H. Covalent modifications of histones affect the packaging of nucleosomal DNA. For example, acetylation of the lysine residues at the N-terminal tails of core histones is believed to loosen the nucleosomal structure and expose the DNA sequence, while deacetylation of these residuesis believed to tighten the nucleosome and make the DNA sequence less accessible (reviewed in Pazin and Kadonaga, 1997 ; Hampsey, 1997). In addition to the compacted chromatin str'uctm‘es, there are many general or gene-specific transcriptional repressor proteins in eukaryotic cells (Hanna Rose and Hansen, 1996). An important flmction of transcriptional activators is to relieve the repression caused by the chromatin structure and/or repressor proteins, an effect often referred to as “anti-repression” or “derepression”. In addition, activators possess the ability to facilitate the intrinsic 51 transcription reaction by acting on the general machinery to increase the efficiency of the transcription process. This is sometimes referred to as “true activation” (Paranjape et al., 1994). Therefore, an activator may firnction both to counteract the repression and to increasethe level of transcription. Many components of the general transcription machinery, including TBP, TFHA, TFHB, TFHF, TFHE, and TFIH-I, have been implicated as direct targets of transcriptional activators (Triezenberg, 1995). Another group of molecules, termed coactivators, mediators or adaptors, may also be involved in gene activation (Pugh and Tjian, 1990; Kelleher et al., 1990; Berger et al., 1990). Transcriptional coactivators are distinct fi'om activators in that most coactivators do not bind DNA directly and none identified so far appears to bind DNA in a sequence-specific manner. Coactivators are distinct fi'om the GT'Fs in that coactivators are dispensable for basal transcription in vitro (Hampsey, 1998). In some cases, coactivators seem to bridge the interaction between gene-specific activator proteins and the general transcription apparatus, while in other cases, coactivators seem to facilitate remodeling of chromatin structure. The interaction between a DNA-bomd activator and a component of the yeast pol H holoenzyme is capable of inducing gene expression in vivo (Barberis et al., 1995; Farrell et al., 1996). Transcriptional Activators Transcriptional activators usually have separable domains for recognizing target genes (DNA-binding domains) and for stimulating the transcriptional machinery (activation domains) (Mitchell and Tjian, 1989; Triezenberg, 1995). The three- dimensional structures of many DNA-binding domains reveal various protein motifs for 52 recognizing specific DNA sequences, such as the helix-turnehelix motif, Zn-fingers, and the leucine-zipper motif (Harrison, 1991). To the contrary, little is known about the structures of transcriptional activation domains. Activators have traditionally been classified according to the most prevalent amino acid of their activation domains, such as acidic, glutamine-rich, proline—rich, or serine/threonine-rich (Triezenberg, 1995). However, mutational analyses indicate that a pattern of bulky hydrophobic amino acid residues may be more important than the more obvious features initially used to distinguish activation domains. Many activators appear to be able to make multiple contacts with various GTFs and coactivators. For example, VP16 has been shown to interact with TBP, TFHB, TFHA, TFIH-I, Ada2, hTAFu32, and dTAFn40 (Barlev et al., 1995; Goodrich et al., 1993; Ingles et al., 1991;1(lemm et al., 1995; Kobayashi et al., 1995; Lin et al., 1991; Xiao et al., 1994). VP16 also interacts with the yeast pol H holoenzyme containing the SRB/mediator complex (Hengartner et al., 1995). This multitude of interaction may reflect the intrinsic complex nature of PIC formation, which involves many protein- protein and protein-DNA interactions. The ability to interact with multiple targets by one activator may synergistically facilitate the assembly of PIC. Alternatively, an activator may interact with different targets at different transitions during transcription. It is also possible that an activator may interact with difl'erent targets at different promoters. Transcriptional Coactivators Transcriptional coactivators function either to bridge the interaction between an enhancer-bound activator and the general transcriptional machinery, or to change the 53 structure of chromatin (Hampsey, 1998; Kaiser and Meisteremst, 1996). These include mammalian USA (upstream stimulatory activity), the SRB/mediator complex, the SWI/SNF and related chromatin-remodeling complexes, and the yeast SAGA and related complexes that catalyze nucleosomal histone acetylation. Although TFHD and TFHA were originally identified as general factors, it is clear that both TFHA and the TAP“ components of TFIH) are not required for basal transcription in vitro and therefore sometimes also classified as coactivators. In addition to these general coactivators that presumably are involved in the activation of many genes, some gene-specific or cell- specific coactivators have also been identified such as OCA-B and T'RAPs (thyroid hormone receptor-associated proteins) (Kim et al., 1996; Fondell et al., 1996; Yuan et al., 1998). The coactivator filnctions of USA, SRB/mediator, chromatin-remodeling complexes, and histone acetyltransferase (HAT) complexes are summarized in the following paragraphs. USA was initially identified in HeLa cells as a fiaction required for transcriptional activation in a pmified in vitro system (Meisteremst et al., 1991). USA includes both positive cofactors (PCs) and negative cofactors (N Cs). Several USA components have been defined, including PCl (poly[ADP-ribose] polymerase), PC2, PC3 (topoisomerase I), and PC4 (Meisteremst et al., 1997; Kretzschmar et al., 1993; Kretzschmar et al., 1994; Merino et al., 1993; Ge and Roeder, 1994b). Among these factors, PC4 binds TBP and several activators and it mediates functional interactions between DNA-bound activators and the PIC (Ge and Roeder, 1994b). Yeast SRB/mediator is a multisubunit complex consisting of Srb2 to Srbl l, Medl to Med4, Med6, Med7, Med8, Gall 1, Sin4, Rgrl, and Rox3 (reviewed in 54 Hampsey, 1998). SRB/mediator, in contrast to TAFus, plays a more general role in transcriptional activation. The SRB/mediator complex is a component of yeast pol H holoenzyme and has several defined activities: 1) stimulation of basal transcription in a highly purified system; 2) response to transcriptional activators in vitro; and 3) stimulation of phosphorylation of the pol H CTD by TFIH-I (Kim et al., 1994; Koleske and Young, 1994). The SRB (suppressor of RNA polymerase B) genes were originally identified in a genetic screen based on suppression of the cold-sensitive growth phenotype associated with trunctions of the pol H CTD, suggesting interactions between the SRB proteins and the CTD (N onet and Yormg, 1989). Several SRB proteins indeed bind the pol H CTD in vitro (Koleske et al., 1992; Thompson et al., 1993). The mediator complex was independently isolated from yeast using a biochemical approach based on its requirement for transcriptional activation by RNA polymerase H in a reconstituted system (Kim et al., 1994). The mediator complex interacts directly with the pol H CTD. Although there are some differences, genetic and biochemical evidence indicates that the SRB complex and the mediator complex likely exist as a single complex present in the pol H holoenzyme (Wilson et al., 1996; Li et al., 1996). This form of pol H holoenzyme is therefore referred to as the SRB/mediator—containing holoenzyme. Not all of the cellular RNA pol H is found in the holoenzyme form as determined by quantitative Western, blots (Kim et al., 1994; Koleske and Young, 1994). It was suggested that the holoenzyme is the form of pol H recruited to most promoters in vivo (Thompson and Young, 1995), although this remains to be firrther tested. Not all genes encoding the SRB/mediator subunits are essential for cell viability and little is known about the mechanisms of their functions. Among these different subunits, Srb2 physically 55 associates with the PIC and binds TBP directly, revealing a functional link between the CTD and TBP (Koleske et al., 1992). Srb5 is a component of the PIC and is required for efficient transcription initiation (Thompson et al., 1993). SrblO and Srbll constitute a kinase/cyclin pair and the SrblO kinase can phosphorylate the pol H CTD (Liao et al., 1995). The phosphorylation of CTD by SrblO prevents formation of the PIC and inhlbits transcription (Hengartner et al., 1998). Gall I, originally identified in a genetic selection for protein factors required for full expression of galactose-inducible genes, copurifies as a SRB/mediator component of the pol H holoenzyme (Kim et al., 1994). Galll enhances basal transcription and facilitates activation by many gene-specific activators. Gall l is shown to interact with TFHE and stimulate the CTD kinase activity of TFIH-I (Sakurai and Fukasawa, 1997 ; Sakurai and Fukasawa, 1998). Several mammalian homologs of yeast SRB/mediator components have been identified and shown to be present in mammalian pol H holoenzymes (Chao et al., 1996; Cho et al., 1998). SWIISNF and related complexes facilitate transcriptional activation by affecting nucleosome structure in an ATP-dependent manner (reviewed in Burns and Peterson, 1997; Kingston et al., 1996). The yeast SWIISNF complex is a multisubunit complex and binds DNA with high-affinity (Cairns et al., 1994; Quinn et al., 1996). Among the SWIISNF submits, Swi2/Snfl is a DNA-dependent ATPase. Swp29 is identical to the ng3 subunit of TFHF and to the TAFn3O subunit of TFHD, implying a link between the SWIISNF, TFHF, and TFHD complexes (Cairns et al., 1996).. The SWIISNF complex was also reported to be a component of the SRB/mediator-containing pol H holoenzyme, however, an independent preparation of pol H holoenzyme does not contain SWI/SNF (Wilson et al., 1996; Li et al., 1996). Although with interesting properties, the SWIISNF 56 complex is only required for activation of several yeast genes, including H0, SUCZ, Ty, ADHI, ADHZ, W0], and STA] (Hampsey, 1998). Many promoters are not dependent on SWI/SNF for activation, indicating that there may be alternative mechanisms for activation fi'om the SWI/SNF-independent genes. Human SWIISNF complex is also capable of altering the nucleosome structure and enhancing the binding of transcriptional activators (Kwon et al., 1994; Imbalzano et al., 1994). Hmnan SWIISNF can also stimulate transcriptional elongation by overcoming the nucleosome-enhanced pausing on the hsp70 gene (Brown et al., 1996). Other chromatin-remodeling factors include Drosophila NURF, CI-IRAC, and yeast RSC complexes. NURF facilitates the GAGA- dependent formation of nuclease hypersensitive sites within a nucleosome array in vitro (Tsukiyama and Wu, 1995). CHRAC facilitates the accessibility of DNA in chromatin and chromatin assembly (V arga-Weisz et al., 1997). RSC (remodels the structures of chromatin) was isolated fi'om yeast on the basis of homology to components of the SWI/SNF complex (Cairns et al., 1996). Similar to SWI/SNF, RSC is a multisubunit complex and has a DNA-dependent ATPase. However, there is yet no evidence that CIRAC or RSC plays a direct role in transcription. Histone acetyltransferases (HATS) catalyze the acetylation of lysine residues at the N-terminal tails of histones. The acetylation of lysine residues neutralizes their positive charge and would presumably reduce the interaction of the core histone tails with DNA, which is postulated to cause gene activation (Paranjape et al., 1994). The direct link between histone acetylation and gene activation was established when the yeast transcriptional coactivator Gcn5 was found to possess HAT activity (Brownell et al., 1996). Gcn5 is a component of the yeast adapter complex, consisting of Ada] , Ada2, 57 Adfi, Gcn5,‘ and Ada5 (Berger et al., 1990; Berger et al., 1992; Candau and Berger, 1996; Horiuchi et al., 1995; Horiuchi et al., 1997; Marcus et al., 1994; Marcus et al., 1996; Pina et al., 1993; Roberts and Winston, 1996). The adapter complex is required for full activation by a subset of transcriptional activators. Both the HAT activity and the interaction with Ada2 were essential for Gcn5 function in vivo (Candau et al., 1997). Recently, several nucleosomal HAT complexes were isolated fi'om yeast (Grant et al., 1997). One of such complexes is named SAGA (Spt-Ada-GcnS-Acetyltransferase) and ' contains Gcn5, Ada2, Spt3, Spt7, Ada5/Spt20 and several TAFns sununits (Grant et al., 1997; Grant et al., 1998). This SAGA complex links nucleosomal histone acetylation with transcriptional activation associated with Ada, TAFn and Spt proteins. The SPT genes were originally identified in a genetic screen for suppressors of a Ty element insertion in the HIS4 promoter (Eisenmann et al., 1989). SPT15 is identical to TBP and many Spt proteins have functions in chromatin dynamics. Similar to SWI/SNF, Gcn5 and Ada proteins are only required for activation from a subset of genes in vivo. Genetic evidence suggests some functional overlap between SWIISNF, SAGA, and SRB/mediator complexes in their roles as coactivators of gene expression (Pollard and Peterson, 1997 ; Roberts and Winston, 1996; Roberts and Winston, 1997). SWI/SNF and SAGA complexes may cooperate to alter the chromatin structure. Alternatively, there may be yet unidentified factors involved in changing the structure of chromatin to facilitate transcriptional activation. It is also possible that the promoter regions of many yeast genes are not tightly packaged into nucleosomes and the alteration of chromatin structure is not essential for formation of functional transcription complexes at these genes. Several HATs newly identified in mammalian cells are also implicated in 58 transcriptional control. These mammalian HATs include human Gcn5, p300/CBP, hTAFu250, P/CAF, and ACTR (Mizzen et al., 1996; Candau et al., 1996; Yang et al., 1996; Chen et al., 1997; Martinez-Balbas et al., 1998). P/CAF exists in a multisubunit complex that also contains some TAFn subunits (Ogryzko et al., 1998). The p300/CBP coactivator, in cooperation with the li gand-activated estrogen receptor, stimulates transcription initiation fiom chromatin templates containing an estrogen response element in vitro (Kraus and Kadonaga, 1998). In addition to using histones as substrates, p300/CBP also acetylates the tumor suppressor protein p53 and enhances its DNA- binding and transcriptional activity (Gu and Roeder, 1997). Another factor, named FACT (facilitate chromatin transcription), was isolated fi'om HeLa cells and moderately stimulates transcript elongation fi'om nucleosomal DNA templates (Orphanides et al., 1998). Although some transcriptional coactivators, such as chromatin-remodeling complexes and HAT complexes, are capable of changing the chromatin structures, the functional relationship between the sequence-specific transcriptional activators and these coactivators is still unclear. If the chromatin structure has to be disturbed before an ' activator can bind its target DNA sequence, how is the chromatin-remodeling complex or HAT initially recruited to a target gene? On the other hand, if an activator recruits the chromatin-remodeling complex or HAT to a target gene, how does the activator bind its target sequence within the compacted nucleosome? Perhaps the chromatin is not a static structure and not all DNA-binding sites are tightly wrapped within nucleosomes. The promoter-recognition by activators and the chromatin-modification by SWIISNF and HAT coactivators may be a cooperative process. An activator may have a limited access 59 to its cognate DNA binding site in the absence of nucleosome alternation or histone acetylation. The binding of the activator to the promoter can then be enhanced by the action of SWIISNF and HAT after they are recruited to the promoter through the activator. The stable association of an activator with the promoter and the alteration cf nucleosome structure then facilitate assembly of the transcription initiation complex. Mechanisms of Activation Transcriptional activators can function by relieving the inhibitory efl‘ect of chromatin and by stimulating the general transcription machinery. In principle, activators can stimulate various steps along the pathway to the synthesis of a mRNA molecule. These include 1) alteration of the chromatin structure; 2) recruitment of GTFs and pol H to a promoter; 3) stimulation of open complex formation; 4) stimulation of promoter escape; 5) stimulation of elongation; and 6) stimulation of reinitiation. Alteration of the Chromatin Structure Chromatin-mediated repression of transcription can be alleviated by the addition of transcriptional activators (Kamakaka et al., 1993; Lorch et al., 1992; Workman et al., 1991). Transcriptional activators may recruit chromatin-remodeling complexes and HATs to specific promoters to alter the local nucleosomal structure and allow assembly of the PIC. It is possible that transcriptional activators work synergistically with chromatin-remodeling complexes and/or HAT complexes to achieve this effect. SWIISNF has been shown to facilitate both activator binding and TBP binding to promoter DNA (Imbalzano et al., 1994; Cote et al., 1994). Many interactions between 60 activators and HATS have also been reported. Among the histone acetyltransferases, TAF 11250 is a subunit of TFHD and has been shown to interact with both activators and basal factors (Ruppert and Tjian, 1995; Wang et al., 1997). CBP and p300 interact with activators including CREB, ElA, and nuclear receptors (Arany et al., 1994; Kwok et al., 1994; Yang et al., 1996; Kamei et al., 1996). Both ACTR and P/CAF also interact with nuclear receptors (Chen et al., 1997). Recruitment of GTFs and pol II The recruitment of GTFs by activators is extensively documented and a large number of activator-GTF interactions have been reported. TFHA, TBP, HB, HE, HF, and [Hi have all been implicated as direct targets for various activators in vitro. The pol H holoenzyme can also be recruited to a promoter by an activator. The recruitment of GTFs and pol H holoenzymes by activators presumably facilitates formation of a functional PIC but may also affect late steps in transcription. Activators such as VP16, Zta, and T antigen bind directly to TFHA, and the binding correlates with their ability to enhance TFHA-TFIHD-TATA complex formation (Damania et al., 1998; Kobayashi et al., 1995; Kobayashi et al., 1998; Ozer et al., 1994). Activators may assist TFHA in overcoming the slow step of TBP binding in PIC formation (Chi and Carey, 1993; Chi and Carey, 1996). The tethering of TBP to a promoter overcomes the requirement for an activator, suggesting that the binding of TBP to a promoter is a slow step that can be accelerated by activators in vivo (Chatterjee and Struhl, 1995; Klages and Strubin, 1995; Majello et al., 1998; Xiao et al., 1995). TBP has been shown to bind in vitro to many activators 61 including VP16, Spl, Oct-l, Oct-2, Gal4, C-Jun, c-Myc, E2F1, and P53 (Ingles et al., 1991; Emili et al., 1994; Zwilling et al., 1994; McEwan et al., 1996; Melcher and Johnston, 1995; Franklin et al., 1995; Pearson and Greenblatt, 1997; Liu et al., 1993; Truant et al., 1993; Chang et al., 1995). Activators can also recruit TFHD through interacting with TAFn subunits. Many activators including VP16, Spl, NTF-l, ElA, progesterone receptor, Bicoid, ERM, ICP4, and estrogen receptor have been shown to bind TAFu subunits in vitro (Goodrich et al., 1993; Klemm et al., 1995; Chen et al., 1994; Gill et al., 1994; Sauer et al., 1995; Schwerk et al., 1995; Defossez et al., 1997; Mazzarelli et al., 1997; Jacq et al., 1994; Carrozza and DeLuca, 1996). T'FHB interacts with several activators such as VP16, Gal4-AH, CTF 1, Gal4, RXRB, CREB, HIV -1 Vpr, and Vitamin D receptor (Agostini et al., 1996; Blanco et al., 1995; Leong et al., 1998; King et al., 1995; Choy and Green, 1993; Kim and Roeder, 1994; Lin et al., 1991; Roberts et al., 1993; Wu et al., 1996; Leong et al., 1998). Activators may induce a comformational change in TFHB which facilitates the binding of TFHB to the TBP-DNA complex (Roberts and Green, 1994; Hayashi et al., 1998). TFIIF interacts with androgen receptor, SRF, and VP16 (McEwan and Gustafsson, 1997; Zhu et al., 1994; Joliot et al., 1995). The interaction between the RAP74 subunit of TFHF and SRF is important for activation by SRF from the c- as promoter (Zhu et al., 1994; Joliot et al., 1995). The activation domain of androgen receptor interacts with both TBP and TFIH", but only the interaction with TFIIF appears to be essential for its activity (McEwan and Gustafsson, 1997). TFHE interacts with Gall 1, a component of the yeast pol H holoenzyme (Sakurai et al., 1996). Genetic evidences suggest that TFIIE and Gall 1 work in a common 62 pathway to regulate transcription in vivo (Sakurai et al., 1997; Sakurai and Fukasawa, 1997). Galll cooperates with TFHE to stimulate the CTD phosphorylation by TFHH in vitro (Sakurai and Fukasawa, 1998). TFHE also interacts with the Epstein-Barr virus activator EBNA 2 through a coactivator p100 (Tong et al., 1995). TFIHi binds VP16, p53, HIV-1 Tat, and RARcr (Xiao et al., 1994; Rochette-Egly et al., 1997; Garcia-Martinez et al., 1997). Both the activation domains of RARa and p53 can be phosphorylated by the kinase activity of TFHH and the phosphorylation correlates with their activities in transcription (Lu et al., 1997; Rochette-Egly et al., 1997). The interaction of Tat with T'FIHi stimulates the phosphorylation of pol H CTD (Garcia-Martinez et al., 1997). The pol H holoenzyme can also be recruited to a promoter through the interaction with activators such as VP16 and Gal4 (Hengartner et al., 1995; Koh et al., 1998). Furthermore, tethering of the holoenzyme to a promoter by fusing a holoenzyme component with a DNA binding domain is sufficient for activation (Farrell et al., 1996). Stimulation of Open Complex Formation The formation of an open complex requires the ATP-dependent DNA helicase activity of TFIH-I. It is possible that binding of activators to TFIHi stimulates its helicase activity and therefore facilitates formation of the open complex. Both VP 1 6 and HIV-1 Tat have been shown to enhance transcription beyond the step of TBP-TATA interaction (White et al., 1992; Xiao et al., 1997). Fmthermore, mutations in the VP16 activation domain have been shown to affect formation of the open complex (Jiang et al., 1994). Activators may stimulate formation of the open complex indirectly through binding 63 TFHE, as TFHE recruits TFIH-I and stimulates its DNA helicase activity. TFIIF was implicated in formation of the open complex by inducing the promoter DNA to wrap around PIC, which may lead to untwisting of the helix near the start site. Transcriptional activators that target TFHF may stimulate the open complex formation by facilitating DNA wrapping. Stimulation of Promoter Escape Promoter escape is an important transition from initiation to productive elongation. This step in vivo is tightly regulated at many promoters. For example, at the Drosophila hsp70 promoter, pol H actively synthesizes short RNA transcript but the transcription complex is stalled near the start site before heat shock. Upon heat shock, the heat shock transcription factor (HSF) stimulates the release of pol H from the promoter and productive elongation begins (O. Brien and Lis, 1991; Rasmussen and Lis, 1993; Lis and Wu, 1993). The CTD of pol H is hypophosphorylated in the stalled complex but highly phosphorylated in the productive elongation complex, suggesting that HSF may stimulate the CTD phosphorylation to stimulate promoter escape (O. Brien et al., 1994). Therefore, by setting up an initiated complex and using the promoter escape as a switch, the cell can respond quickly to stress signals such as heat. Stimulation of Elongation Although many activators stimulate initiation, some appear to primarily stimulate elongatiOn (Blau et al., 1996). Transcriptional activators VP16, ElA, HIV-1 Tat, p53, and E2F1 have all been shown to stimulate elongation (Blau et al., 1996; Yankulov et al., 1994). Activators may recruit elongation factors to the transcription complex and affect both the rate and processivity of the elongation complex. For example, HIV-1 Tat stimulates transcription of the HIV-1 viral genome primarily by increasing the processivity of pol H, presumably by stimulating the phosphorylation of pol H CTD (Garcia-Martinez et al., 1997; Parada and Roeder, 1996). Several activators including SRF and androgen receptor interact directly with TFHF and may enhance transcript elongation. Activators may also target other known elongation factors such as P-TEFb, SH, SHI, and ELL. Stimulation of Reinitiation DNA-bound activators may hold certain initiation factors at the promoter when pol H begins elongation. This would facilitate the subsequent assembly of an initiation complex and stimulate multiple rounds of transcription. Both Gal4-VP16 and Oct-2 remain bound to the DNA binding sites after initiation and stimulate reinitiation from the templates (White et al., 1992; Amosti et al., 1993). The ligand-bmmd estrogen receptor tightly associates with its cognate DNA site and stimulates reinitiation by promoting the reassembly of the preinitiation complex (Kraus and Kadonaga, 1998). The CTD phosphatase may be targeted by some transcriptional activators to regulate the pool of pol HA, which is important for transcriptional reinitiation. TRANSCRIPTIONAL REPRESSION In principle, transcriptional repression is equally important as activation for appropriate gene expression in vivo (reviewed in Clark and Docherty, 1993; Hanna Rose 65 and Hansen, 1996). Both gene-specific repressors, which inhibit the transcription from specific promoters, and general repressors, which inhibit the transcription fi'om multiple and unrelated promoters, have been identified. The repression of transcription can result from competitive binding between a repressor and an activator, squelching of a GTF or a cofactor necessary for activation, direct targeting of the basal machinery by a repressor, or alteration of the chromatin structure. Several repressors appear to direcfly affect the TBP-TATA interaction. Motl, originally identified in yeast, inhibits the binding of TBP to the TATA sequence in an ATP-dependent manner (Auble and Hahn, 1993; Auble et al., 1994). The inhibitory efl‘ect can be overcome by the addition of TFHA or TFHB. Motl is a member of the swi2/snfl family of ATPase, but the Motl ATPase activity is not stimulated by DNA (Auble et al., 1997). Although functioning as a repressor on some promoters, Motl can also flmction as a coactivator on other promoters presumably by removing TBP fi'om nonfirnctional TATA sequences (Collart, 1996; Madison and Winston, 1997). The Not (negative on TATA) complex from yeast are global repressors that target the general transcriptional machinery and preferentially affects basal, rather than activated, transcription (Collart and Struhl, 1994). While mot] mutations decrease transcription fi'om TATA-less promoters, not mutations increase transcription from TATA-less promoters (Collart, 1996). Genetic evidence suggests that NOT proteins negatively regulate the activity of factors such as Spt3 and TFHA that promote TBP- TATA interaction. NOT proteins, Motl , Spt3, and TFHA may functionally interact to regulate the distribution of TBP on strong and weak promoters. 66 The human Drl -DRAP1IN C2 complex represses transcription by blocking the association of TBP with TFHA and TFIIB (Inostroza et al., 1992; Meisteremst and Roeder, 1991). Both Drl and DRAPl proteins contain histone-fold motifs that appear to mediate the interaction between these two submits (Mermelstein et al., 1996). Drl also interacts directly with TBP (Meisteremst and Roeder, 1991; Inostroza et al., 1992). Histone deacetylases (HDAs) are also implicated in repressing transcription. A direct link between histone deacetylation and transcriptional repression was established when the human histone deacetylase I-H)A was found to be homologous to yeast de3, a known transcriptional repressor identified in a genetic screen (Talmton et al., 1996; Vidal and Gaber, 1991). Besides de3, several other histone deacetylases have also been identified in yeast, suggesting a possible flmctional redundancy (Carmen et al., 1996; Rundlett et al., 1996). The yeast histone deacetylase de3 exists in a multisubunit complex containing corepressor Sin3 and other polypeptides (Kasten et al., 1997). Large HDA complexes containing de3 and Sin3 homologs have also been identified in mammalian cells and they are implicated in mediating repression by unliganded nuclear hormone receptors and by Mad-Max, in each case dependent upon med3 and mSin3 ( Alland et al., 1997; Hassig et al., 1997; Hassig et al., 1998; Heinzel et al., 1997 ; Laherty et al., 1997; Nagy et al., 1997; Zhang et al., 1997). It was proposed that the replacement of the IHDA complex with a HAT complex, upon either receptor-hormone binding or removal of Mad-Max by Myc-Max, switches the transcriptional repression to activation. Certainly, more thorough and insightful experiments have to be carried out to generate a clear view on how transcriptional regulators fimction through altering the chromatin structure. 67 OVERVIEW Human TFHF is an 0.sz tetramer of RAP30 and RAP74 subunits. It was previously known that TFHF plays important roles in both transcriptional initiation and elongation. It was suggested that T'FHF may also be involved in transcriptional recycling based on its interaction with pol H. However, the mechanisms of TFHF in these distinct steps were unclear. One primary flmction of TFHF is to escort pol H to the PIC. Both RAP30 and RAP74 are essential for forming a stable DBpolF complex. RAP30 binds RAP74, TFHB, and pol H in vitro. RAP30 shares sequence similarities with bacterial 0' factors. The central region of RAP30 exhibits significant sequence similarity with the polymerase- binding domains of 07° and 043. The C-terminal region of RAP30 has a cryptic DNA- binding domain similar to the conserved region 4 of bacterial 0 factors. The NMR structme of the DNA-binding domain of RAP30 reveals its similarity with histone H5 and other members of the “winged” helix-turn-helix family. Mutational analyses demonstrated that transcriptional activities of RAP30 are mediated by separable domains. It was shown that the pol H-binding region of RAP30 is only necessary for elongation, while the cryptic DNA-binding domain of RAP30 is only necessary for initiation. The region of RAP30 necessary for association with RAP74 is essential for both initiation and elongation. After the cDNA encoding human RAP74 was cloned, a number of RAP74 deletion mutants were generated in our laboratory. It was shown that the N-terrninal domain of RAP74 direcfly binds RAP30, while the C-terminal domain of RAP74 binds 68 both TFHB and pol H. The binding of RAP74 to TFHB prevented the interaction between TFHB and RAP30, indicating dynamic interactions between TFHF and TFHB. The C-terminal domain of RAP74 also stimulated the dephosphorylation of the pol H CTD by a CTD phosphatase. The N-terminal domain of RAP74 appeared to be critical for transcription, although the deletion of C-terminal regions moderately affects initiation and elongation. RAP74 can be phosphorylated by protein kinases such as casein kinase H, TAFn250, and TFIH-I. RAP74 also interacts with TFHEa and transcriptional activators including SRF, androgen receptor, and VP16. The interaction with RAP74 is important for the SRF-mediated activation of the c-fos promoter. My research project focuses on the functions and mechanisms of RAP74 in transcription by pol H. I developed several assays to dissect the functional domains of RAP74. In Chapter 2, I demonstrate that the N-terminal domain of RAP74 is necessary and sufficient to support both preinitiation complex formation, initiation and elongation. The same region is also critical for inducing a tight DNA wrap around pol H in the preinitiation complex as detected by our collaborators using photocrosslinking techniques. These novel findings, along with evidence in the literature, lead to the model that the DNA bending and wrapping are important in both transcriptional initiation and elongation. The analysis of RAP74 deletion mutants indicates that a small region, amino acids 136 to 217, of RAP74 is critical for DNA wrapping, initiation, and elongation. This region of RAP74 is highly conserved and may be important for formation of the TFHF tetramer. In Chapter 3, I describe a series of RAP74 proteins with site-directed mutations introduced in this critical region. Using a highly sensitive assay, I analyzed the activities of these mutant proteins in elongation and calculated the average elongation rate for each 69 TFHF mutant. Several amino acid residues in RAP7 4 were found to be critical for stimulating the elongation rate of pol H. It was previously suggested. that TFHF stimulates the elongation rate by suppressing the transient pausing of pol H. Comparing TFIIF containing wild type RAP74 or RAP74 mutants, it is clear that TFHF does not change the positions at which pol H pauses, but T'FHF affects the dwell time of pol H at each nucleotide position. The activities of RAP74 mutants in elongation match very well with their activities in initiation, further strengthening the notion that, in contrast to RAP30, RAP74 has an identical role in both initiation and elongation. The capacity of these RAP74 point mutants in wrapping DNA around pol H in the PIC remain to be tested. 1 was puzzled by the finding that both the central region and the C-terminal domain of RAP74 are not required for either initiation or elongation, particularly because these regions are involved in many protein-protein interactions and the C-terminal domain of RAP74 is highly conserved. In Chapter 4, I analyze RAP74 deletion mutants in the multiple-round assay and the single-round assay. It is clear that the C-terminal domain and the central region of RAP74 are required for continuing initiation at later reaction times, although both regions are dispensable for the first-round of transcription. This is the first direct evidence that TFHF is involved in transcriptional reinitiation. Using a G-less cassette, I further demonstrate that transcription reinitiation in the extract system is not limited by the physical amount of DNA template or pol H molecules; rather, a kinetic event, possibly the conversion of pol HO to pol HA, limits the capacity of the system to reinitiate. This model is consistent with the finding that the C-terminal domain of RAP74 stimulates the conversion of pol HO to pol HA by a CTD phosphatase. 70 In Chapter 5, I test the activities of other general transcription factors in elongation assays. As predicted by the DNA wrapping model, TBP, TFHA, TFHB, and TFIIE cooperate to stimulate the elongation rate in a TFHF-dependent manner. This observation suggests that these factors may indeed work in concert with TFHF to wrap DNA template around pol H during elongation. The results of my research project provide many insights into the functions of RAP74, and the general mechanisms of initiation and elongation. Several assays are well developed and should be very useful in the future study of TFHF. Directions for future study are discussed. 71 CHAPTER 2 FUNCTIONS OF THE N-TERMINAL DOMAIN OF RAP74 IN INITIATION AND ELONGATION HVTRODUCTION RNA polymerase H (pol II) interacts with a number of general and regulatory factors to initiate transcription accurately from a promoter (reviewed in Orphanides et al., 1996; Zawel and Reinberg, 1993). In the pathway toward initiation, promoter DNA is bent, and DNA may be wrapped around pol H (Kim et al., 1997; Robert et al., 1998). General factors TBP (or TFHD), TFHB, TFHF, and TFHE cooperate with pol H to strain the DNA helix arormd the transcriptional start site before ATP-driven helix opening by TFIH-I (Orphanides et al., 1996; Zawel and Reinberg, 1993). After initiation, pol H releases fi'om the promoter (promoter clearance or promoter escape), elongates the RNA chain, terminates transcription, and recycles. TFHF, made up of RAP30 (RNA polymerase H-associating protein 30 kDa) and RAP74 (58 kDa) sublmits, may participate in each of these stages of the transcription cycle. Inspection of its 517 amino acid (a) sequence indicates that human RAP74 can be divided into three regions: 1) a highly basic N-terminal domain with significant globular structure (aa 1-217); 2) an overall acidic, highly charged central region lacking in hydrophobic amino acids but rich in E, D, K, R, S, T, G, and P (a 218-398); and 3) a very basic C-terminal domain with globular structure (a 399L517) (Aso et al., 1992; Finkelstein et al., 1992). The N-terminal domain is important for RAP30 binding (Wang and Bmton, 1995; Yonaha et al., 1993), preinitiation complex assembly (this chapter), and elongation stimulation (Kephart et al., 1994; this chapter). The C-terminal domain of 72 RAP74 makes contact with TFHB (Fang and Burton, 1996) and pol 11 (Wang and Burton, 1995) and stimulates the activity of a pol H carboxy-terminal domain (CTD) phosphatase that may have roles in initiation, elongation, termination, and recycling (Chambers et al., 1995) A pathway has been defined for assembly of preinitiation complexes on TATA box-containing promoters (Orphanides et al., 1996; Zawel and Reinberg, 1993). The TATA-binding protein (TBP) subunit of TFHD binds to the TATA sequence. Insertion of TBP into the DNA minor groove at TATA induces a 950 bend (Kim et al., 1993; Kim et al., 1993). TFHB can then enter to form the DB complex, made up of TBP (or TFHD), TFHB, and promoter DNA. The C-terminal repeats of TFHB bind DNA upstream and downstream of TATA, stabilizing the DNA bend (Lagrange et al., 1996; Nikolov et al., 1995). The N—terminal domain of TFIIB may extend toward the transcriptional start site as a scaffold on which to assemble pol H and TFHF (Orphanides et al., 1996). To bind efficienfly to the promoter, pol H must first bind TFHF (Conaway et al., 1991; Flores et a1.,l991;Killeen et al.,1992). In some cases, the RAP30 subunit has been sufficient to deliver pol H to the promoter (Flores et al., 1991; Killeen et al., 1992; Tyree et al., 1993), but the RAP74 subunit contributes to proper assembly, complex stability, and initiation. For promoters with weak TATA boxes, both RAP30 and RAP7 4 contribute to template commitment of TFHD, TFHB, and pol H (Tan et al., 1994). Fmthermore, RAP74 strongly stimulates initiation fiom supercoiled and pro-melted templates that are dependent only on TBP, TFIIB, pol H, and TFHF for accurate transcription (Pan and Greenblatt, 1994; Parvin et al., 1994). In most contexts, therefore, 73 both the RAP30 and RAP74 subunits are important for TFHF firnction in complex assembly and initiation. After fulfilling its role in initiation, TFHF stimulates the elongation rate of pol H (Bengal et al., 1991; Izban and Luse, 1992; Kephart et al., 1994; Price et al., 1989; Tan et al., 1994). On non-chromatin DNA templates and in the absence of other general factors, TFHF can accelerate polymerization to up to 1,500 nucleotides per min, close to the estimated physiological rate (Izban and Luse, 1992a). TFIIF suppresses pausing by pol II (Bengal et al., 1991; Izban and Luse, 1992a; Price et al., 1989), but whether this is a cause or effect of rate stimulation is not known. Both the RAP30 and RAP74 subunits of TFIIF are required for elongation stimulation (Kephart et al., 1994; Tan et al., 1994), and preliminary mapping studies indicate that the N-terminal domain of RAP74 is most important for elongation (Kephart et al., 1994). Tan et al. (Tan et al., 1995) identified a class of RAP30 mutants that are impaired for both elongation stimulation and accurate initiation, and these mutants are also defective for binding RAP74, consistent with the requirement of both subunits for elongation. They have also identified classes of RAP30 mutants that are defective only for elongation stimulation or for initiation, but not for both. In' contrast to their results with RAP30, however, we find that a region within the N-terminal domain of RAP74, that is not essential for RAP30 binding, is nonetheless strongly stimulatory for both initiation and elongation. 74 Materials and Methods Transcription factors and extracts Recombinant Saccharomyces cerevisiae TBP and human recombinant TFHB were the kind gifts of Steven Triezenberg and Fan Shen. The clone for production of TFHB was the kind gift of Danny Reinberg. Recombinant human RAP30, RAP74, and RAP74 mutants were prepared and quantitated as described (Fang and Burton, 1996; Wang et al., 1993; Wang et al., 1994; Wang and Burton, 1995). Construction of new mutants is described below. Calf thymus pol H used in electrophoretic mobility shift experiments was prepared by the method of Hodo and Blatti (Hodo and Blatti, 197 7) and was primarily in the Hb form, lacking the carboxy terminal domain (CTD). Gel mobility shift experiments with calf thymus pol Ha (the kind gift of Richard Burgess) gave similar results (data not shown). Human HeLa cells were purchased from the National Cell Culture Center (Minneapolis, MN). Extracts of HeLa cell nuclei were prepared as described (Shapiro et al., 1988). A TFHF-depleted extract was prepared by immunoprecipitation of TFHF with anti-RAP30 and anti-RAP74 antibodies (Burton et al., 1988; Finkelstein et al., 1992). The TFHF-depleted extract was completely dependent on the re-addition of RAP30 for activity and was strongly stimulated by addition of RAP74. Construction of RAP74 mutants RAP74(l-217) was constructed by polymerase chain reaction amplification of a plasmid clone encoding RAP74 with primers 5'-CATATGGCGGCCCTAGGCCCT-3' and 5'-C1‘CGAGAGACATTTCCAGGT-3' and subcloning between the Mid and XhoI sites (cloning sites are underlined) of pET2 1 a (N ovagen). Three triple-alanine mutations 75 were constructed in RAP74(1-217), using the Quick Change Site-Directed Mutagenesis Kit (Stratagene) and appropriate primers. These mutant proteins are named RAP74(1- 217)170A3, 173A3, and 176A3. Each of these mutant proteins has the sequence AAA beginning at the indicated amino acid, so, for example, 170A3 carries V170A, L171A, and N172A (Fig. 5). Mutated RAP74 genes were confirmed by DNA sequencing. Electrophoretic mobility shift assay The DNA probe for the gel Shift assay was the adenovirus major late promoter from position -53 to +14 relative to the transcriptional start site. The probe was synthesized by the polymerase chain reaction using the primers: 9-32p- CAGGTGT'I‘CCTGAAGG-3' and 5'-ATGCGGAAGAGAGTGA-3'. The template for PCR is pML containing the wild type adenovirus major late promoter (-258 to +196). The upstream primer was radiolabeled using y-32P-AT'P and T4 polynucleotide kinase. After amplification, the DNA probe was gel-purified using the Qiaex kit (Qiagen). Mobility Shifts were performed according to Wang and Burton (Wang and Burton, 1995) with some modifications. The reaction mixtm'es (15 ul) contained 20 mM HEPES pH 7.9, 20 mM Tris pH 7.9, 50 mM KC], 2 mM DTT, 0.5‘ mg/ml BSA (bovine serum albumin), 10% v/v glycerol, radiolabeled DNA probe, and proteins, incubated at 30 0c. Saccharomyces cerevisiae TBP (0.3 pmol) was combined with the DNA probe (approximately 40 finol) for 15 min. Recombinant human TFHB (0.3 pmol) was then added and incubated for 15 min. Calf thymus pol H (0.15 pmol) was incubated with human recombinant TFHF (0.1 pmol) for at least 5 min prior to addition to the DB complex. For reactions involving separate TFHF subunits, pol H was incubated with RAP30 for 5 min and then mixed with RAP74 or a RAP74 mutant and preincubated for 76 an additional 5 min before addition to DB and further incubation for 15 min. It appeared that prior addition of RAP30 to pol H aided assembly of RAP74 into DBPolF. Reaction mixtln'es were loaded onto a 4% polyacrylamide gel containing 0.09 % bisacrylamide, 2.5 % glycerol and 0.5X TBE (triS-borate-EDTA). Dried gels were analyzed by autoradiography. Transgmtion assays Immobilized tem lates Preparation of immobilized templates was adapted fiom published methods (Arias and Dynan, 1989; Marshall and Price, 1992). DNA containing the adenovirus major late promoter was synthesized by the polymerase chain reaction using an upstream 5'- biotinylated primer. The template for amplification was a pBluescript H SK(-) vector (Stratagene) containing the adenovirus major late promoter (-258 to +196) subcloned between the XhoI and HindIH Sites of the plasmid. The sequence of the upstream primer was 5'-biotin-CCCI‘CGAGCGGTGTI‘CCGCGGTCCI‘CCTCG-3', and the sequence of the downstream primer was 5'-CGGTGGCGGCCGCTCTAGAACTAGTGGATC-3'. The template extended fi'om positions -263 to +251. Biotinylated DNA was incubated with streptavidin paramagnetic beads (CPG) in 2 M NaCl, 1 mM EDTA, 10 mM Tris- HCl, pH 7.5 for 15 min at room temperature. Immobilized templates were collected with a magnetic particle separator (CPG), washed 4 times and stored at 4°C in phosphate buffered saline pH 7.2 containing 1 mg/ml BSA and 0.03% NaN 3. Pulse-spin and pulse-chase single-round assays A 20 ul reaction mixtlue consisted of 2 ul paramagnetic beads (about 0.6 ug DNA) carrying the adenovirus major late promoter and TFHF—depleted transcription 77 extract (72 ug total protein) supplemented with recombinant RAP30 and RAP74 or a RAP74 mutant (10 pmol each), in transcription buffer (12 mM HEPES, pH 7.4, 12% glycerol, 0.12 mM EDTA, 0.12 mM EGTA, 1.2 mM DTT) containing 60 mM KCl and 12 mM MgC12. Preinitiation complexes were formed for 60 min at 30 0C. 100 uM ATP, CT'P, GTP and 1 uM a32P-UTP (10 poi per reaction) were added to initiate transcription for 1 min. For the pulse-spin protocol, template-associated complexes were diluted with 200 ul transcription buffer containing 60 mM KCl and 1 mg/ml BSA, isolated by centrifugation, and extracted fi'om beads by boiling in 20 ul 9o % v/v formamide, l % SDS, 10 mM Tris pH 7.9, 1 mM EDTA, 0.01 % bromophenol blue and 0.01 % xylene cyanol. For the pulse-chase protocol, instead of diluting and centrifuging samples, 1 mM ATP, UTP, GTP, and CT? were added 1 min after addition of NTPS, and elongation continued for 10 min. Reactions were stopped by addition of 200 ul 0.1 M sodium acetate pH 5.4, 0.5 % SDS, 2 mM EDTA, and 100 ug/ml tRNA, followed by phenol- chloroform extraction and ethanol precipitation. Samples were electrophoresed in a 10 % polyacrylamide gel containing 1X TBE and 50 % w/v urea. For quantitation of the gel, the signal in the presence of RAP30 and the absence of added RAP74 was used to estimate background (Figure 2, lanes 21 and 22). The weak signal obtained in the absence of added RAP74 was attributed to residual RAP74 remaining in the TFHF- depleted extract. Elongation stimulation assay Stimulation of pol H elongation was determined by adding TFIIF subunits to transcriptionally engaged pol H molecules that were washed fi'ee of associated elongation factors. A 20 ul reaction mixture consisted of 2 ul paramagnetic beads carrying the 78 adenovirus major late promoter, transcription extract derived from HeLa ceH nuclei (108 ug total protein), and transcription bufl‘er containing 60 mM KCl and 12 mM MgC12. Samples were incubated 60 min at 30 0C to form preinitiation complexes. Transcription was initiated by addition of 100 uM AT'P, GTP, CTP, and 1 uM (10 uCi) ct-32P-UT'P (2 ul volmne). After 1 min, elongation complexes were diluted in 200 ul transcription bufl'er containing 500 mM KCl and 1 mg/ml BSA and isolated by centrifugation. The prupose of this treatment was to remove accessory factors fiom the elongation complex. The 500 mM KCl wash appeared to be effective because elongation rate stimulation was highly dependent on addition of both RAP30 and RAP74 (Fig. 3). Complexes were diluted with transcription buffer containing 60 mM KCl and 1 mg/ml BSA and isolated by centrifugation two times. Complexes were then resuspended in 20 ul transcription buffer containing 60 mM KCl and 12 mM MgC12, recombinant RAP30 and RAP74 or a RAP74 mutant were added (10 pmol each) and incubated for 5 min. 100 uM ATP, GTP, CTP, and UTP were added in 2 ul, and transcripts were elongated for 2 min. Transcripts were isolated by phenol extraction and ethanol precipitation and electrophoresed in a 6% polyacrylamide gel, as described above. Accurate transcription was quantitated for all of the transcripts from +122 to +251. Elongation stimulation was determined by subtracting the average value for samples containing RAP30 but no RAP74 as background (Fig. 3A, lanes 14‘and 15) and expressed as percent of the highest signal obtained for RAP74 (lane 3). 79 RESULTS The N -terminal domain of RAP74 supmrts preinitiation complex assemny A primary firnction of TFIIF in accurate initiation is to deliver pol H to the promoter, so we used an electrophoresis mobility shift assay to determine which regions of RAP74 were required to bring pol H into a stable complex with adenovirus major late promoter DNA, TBP (D), TFHB, and RAP30 (DBPolF complex) (Fig. 1). By itself, TBP did not efficiently induce a Shift of the promoter fi'agment (lane 2). Upon addition of TFHB, however, a DB complex consisting of TBP, TFHB, and promoter DNA was observed (lane 3). When pol H was added to DB, a weak DBPol Shift was seen (lane 4). RAP30 alone did not stimulate pol H binding to DB (lane 6), nor did RAP74 (data not shown). . The TFHF complex and separately added RAP30 and RAP74 subunits, however, supported assembly of DBPolF (lanes 5 and 7). RAP74(1-172) was minimally required to support assembly (lane 10). A number of RAP74 mutants that have been Shown to be defective for RAP30 binding (Wang and Burton, 1995; Yonaha et al., 1993) failed to support formation of DBPolF (lanes 11-15). The different mobilities of complexes containing RAP74 deletion mutants may be attributable to differences in the charge or the degree of DNA bending or flexibility in the complex. Because RAP74(1- 517), (1-296), (1-205), and (1-172) are predicted to carry charges of 0, -4, +6, and +7, however, it is difficult to account for all observed mobility differences solely on the basis of charge. For instance, RAP74(1-172) is predicted to be more basic than RAP74(1-205) and yet supported a DBPolF complex with a faster mobility. Furthermore, RAP74(1- 205) and (1-172) have different transcriptional activities (see below). 80 Figure 1. An electrophoretic mobility shifi assay was used to analyze the requirement for RAP74 to form DBPolF. The probe was the adenovirus major late promoter between positions -53 and +14. Key: D) recombinant yeast TBP (0.3 pmol); B) recombinant human TFIIB (0.3 pmol); Pol) calf thymus pol II (0.15 pmol); F) recombinant human TFIIF complex or RAP30 and RAP74 or a RAP74 mutant, added separately (0.1 pmol). DBPolF* indicates the different mobilities of complexes containing different RAP74 mutants. 81 Awmmroflsveivm Esswiém Emivfiém 5-32am 62-34;; Stevenson Goméfiém GmNéEEM Eraser; 0m vtom ++++++++++++ +++++++++++++ ++++++++++++++ 2 3 4 5 6 73891101112131415 Pol D 1 Figure l 82 Tyree et al. (Tyree et al., 1993) reported DBPolFBO and DBPolF74 complexes, which formed with only the RAP30 or the RAP74 subunit of TFHF, but these investigators used a different promoter and Drosophila TBP, TFHB, pol H and human TFHF, rather than yeast TBP, human TFHB, human TFHF, and bovine pol H, as used in this study. Killeen et al. (Killeen et al., 1992) and Flores et al. (Flores et al., 1991) also reported DBPol30 and DABPol30 complexes using very different buffer conditions from those used here. Although RAP74 was not essential for assembly in those studies, it was strongly stimulatory. This is the first report that Shows the minimal region of RAP74 that stimulates incorporation of pol H into DBPolF. The N-terminal domain of RAP74 supmrts accurate initiation To map the flmctional domains of RAP74 important in transcription, a reconstitution assay was set up using recombinant TFHF proteins and TFIIF-depleted HeLa nuclear extracts (Fig. 2). Native TFHF proteins were depleted from the HeLa nuclear extracts by immunoprecipitation with or-RAP30 and a-RAP74 antibodies. The depleted extract was completely inactive in transcription (compare lanes 3 and 2, Fig. 2A), and the addition of recombinant RAP30 and RAP74 supported accru'ate transcription (lanes 6-9, Fig. 2A). The activity observed with the addition of recombinant RAP30 was attributed to the residual amount of native RAP74 in the depleted extract (lane 4). When a saturating amount of RAP30 was used, the level of transcription saturated in the presence of 2 pmol of RAP74 (lanes 3-6, Fig. 2B). . To determine which regions of RAP74 are most important for initiation, RAP74 mutants were tested for accurate initiation and runoff transcription from the adenovirus major late promoter (Fig. 3). RNA within the early elongation complex was visualized 83 Figure 2. Accurate initiation from an adenovirus major late promoter in the HeLa extract is dependent on both RAP30 and RAP74 subunits of TFIIF. A TFIIF-depleted extract was prepared by immunochromatography with ct-RAP30 and (ll-RAP74 antibody columns, and concentrated by amonium sulfate (AMS) precipitation. A) Reactions (20 ul each) contained 800 ng pML digested with SmaI, 4 ul HeLa nuclear extracts (N E, lanes 1 and 2), or various amounts of TFIIF-depleted extracts as indicated (DE, lanes 3-9) supplemented with 10 pmol recombinant human RAP30 (lane 4), or RAP74 (lane 5), or RAP30/RAP74 complex (lanes 6-9), in transcription buffer containing 60 mM KCl and 12 mM MgC12. In lane 1, 200 ng or-amanitin was also included. After 60 min incubation, 600 uM ATP, GTP, CTP, and 25 uM a-32P-UTP (10 uCi per reaction) were added and reactions continued for 30 min at 30 0C. The reactions were stopped and RNA transcripts were isolated and analyzed on a 6% polyacrylamide gel containing 50% urea. B) Similar to A, except increasing amounts of recombinant RAP74 (0, l, 2, 5 pmol) were added in lanes 3-6. 10 pmol recombinant RAP30 was added in lanes 3-6. Reactions also contained either 4 111 NE (lane 1) or 6 111 DE (lanes 2-6). 84 or-30 or-74 AMS HeLa Cells —>HeLaNE -—> FT ——->FT ——> DE columns columns prec. A (xA + 30 + 74 + 30/74 + + + + DE 4 4 4 2 4 8 10 NE + + 1 2 3 4 5 6 7 8 9 B NE + DE + 30 74 85 Figure 3. Regions of RAP74 required for accurate initiation. A) Short and runoff RNAS accurately initiated from the adenovirus major late promoter. All lanes contained TFIIF- depleted extract (DE; 72 pg total protein), recombinant human RAP30 (10 pmol), and RAP74 or a RAP74 mutant (10 pmol), preincubated with immobilized template for 60 min. Transcripts were initiated with all four NTPS and radiolabeled with a32P-UTP (ACGU*) for l min. For the pulse-chase protocol, samples were chased by addition of 1 mM each NTP for 10 min (+). For the pulse-spin protocol, initiated complexes were diluted with transcription buffer and centrifirged briefly to isolate Short, template- associated RNAS (-). The approximate sizes of short RNAS can be estimated by comparison to 5'-phosphorylated 16 and 18 nucleotide (nt) DNA markers. In lane 1, AMPPCP (a B-y non-hydrolyzable ATP analogue) and 2'-3' dideoxy ATP (ddA) were substituted for ATP. In lane 2, AMPPCP was substituted for ATP. In lane 3, 1 jig/ml ct- amanitin was included in the reaction. The gel band indicated with an asterisk is not a pol II transcript because it is synthesized in the presence of l rig/ml or-amanitin (data not shown). B) Phosphorimager quantitation of the data shown in "A" combined with data from two other experiments, reported as average +/- standard deviation. Short transcripts generated in the presence of RAP74 are expressed as 100%. 86 m enema 9£l'l ZLI‘I SOZ'I LIZ‘I 96Z'I 9991 60V'I LIS'I u 0 U 9 (%) uouduosuml 035-83g D samba—am I is m Essa co ES. cambmie 2.2m . _~ +251—-) +192—> M m m a 5 3 2% a :5 o l’9 ‘7' l\ l\ l‘ q —t —t —l r—t —.t 12345678910111213 I IWi! Illl n I... T when. - Ills _ so... 0! 14 15 16 17 18 19 20 21 22 23 24 25 26 FigureS 96 cuou £V9Ll EVELI I I ll m 0.53m SVOL I L i it gamma—o I 830-023 D LIZ‘I LIS'I (%) "Olldposusrl 97 DISCUSSION From primary sequence (Aso et al., 1992; Finkelstein et al., 1992), RAP74 is proposed to have highly basic N- and C-terminal domains separated by a highly charged, overall acidic, and flexible central region that is rich in charged amino acids, E, D, K, and R, and also S, T, P, and G. In this chapter, we Show that these primary sequence features correspond to distinct N- and C-terminal fimctional domains. The N-terminal domain is suficient to support preinitiation complex assembly, single-round initiation, and elongation by pol H. The RAP74 N-terminal domain extending fi'om aa 1-217 supports most RAP74 functions for preinitiation complex assembly (Fig. 1), accurate initiation (Fig. 3), and elongation stimulation (Figs. 4 and 5). In this work, we have mapped a critical region for these functions between a 136-217. RAP74(1-172) binds tightly to the RAP30 subunit, but RAP74(1-136) does not (Wang and Burton, 1995; Yonaha et al., 1993). Consistent with its capacity to bind RAP30, RAP74(1-172) is minimally suficient to support pol H assembly into DBPolF (Fig. 1). Fulfilling this role in assembly, however, is not sufficient to support TFHF function in initiation or elongation, which minimally requires RAP74(1- 205) (Fig. 3). RAP74(1-217) is Significantly more active than RAP74(1-205) in both accurate initiation and elongation stimulation (Figs. 3 and 4). Recent site-specific DNA photocrosslinking studies Show that RAP74 induces a significant conformational change within the DBPolFE preinitiation complex (Forget et al., 1997 ; Robert et al., 1996; Robert et al., 1998). This conformational change is referred to as “isomerization” to compare it with isomerization of the E. coli preinitiation complex, which involves Similar changes in protein conformation (Roe et al., 1985) and 98 wrapping of promoter DNA around RNA polymerase (Craig et al., 1995; Polyakov et al., 1995). Interestingly, RAP74(1-205), which iS minimally required for accurate initiation, also minimally supports isomerization of DBPolFE as indicated by development of a nlnnber of specific DNA photocrosslinks with the RPBl and RPB2 subunits of pol H, with RAP30, and with TFHE34 (Forget et al., 1997; Robert et al., 1996; Robert et al., 1998). Because RAP74(1-172) is sufficient for tight RAP30 binding and DBPolF assembly but insufficient for isomerization of DBPolFE, accurate initiation or elongation stimulation, RAP74(1-217) and (1-205) appear to have functions that are not communicated to pol H directly through the RAP30 subunit. Photocrosslinking studies have indicated that RAP74 approaches the adenovirus major late promoter at numerous positions extending all the way from —56/-6l upstream of TATA to +26 downstream of +1 (about 280 angstroms of B-form DNA) (Robert et al., 1998; Forget et al., 1997). This extensive crosslinking “footprint” is the basis for one argument in favor of DNA wrapping around pol H in DBPolFE and also for an (12132 heterotetrameric form of TFIIF in the commex (Robert et al., 1998). Because RAP74 interacts with promoter DNA and induces isomerization of DBPolFE, RAP74 appears to support DNA wrapping both by contacting DNA directly and by modifying the contacts of RAP30, pol H, and TFHE34 with DNA (Robert et al., 1998; Robert et al., 1996; Forget et al., 1997). The region of RAP74 between a 172-205, therefore, appears to stimulate transcription by helping DNA to wrap around pol H. Wrapping may result from direct interactions between DNA and aa 172-205 or this region may be involved in protein-protein interactions that facilitate wrapping. Recent work indicates that aa 172-205 is involved in dimerization of RAP74 (Robert et al., 1998). It is not clear whether the adjacent region of RAP74 fi'om 99 aa 205-217, which also stimulated initiation and elongation, contributed to DNA wrappinglor another function. It iS interesting to note that DBPolF complexes containing RAP74(1-517), (1-296), (1-205), and (1-172) had different electrophoretic mobilities that could have been caused by differences in the degree of DNA bending or flexibility (Fig. l). Difl‘erences in mobility might relate to the degree of DNA bending caused by partial or complete isomerization of DBPolFE in the presence of TFHF mutants. RAP74(1-217), (1-205), and (1-172) Showed a spectrum of decreasing activities in both accurate initiation and elongation stimulation (Fig. 4C). Triple alanine mutants in this region, RAP74(1-217)170A3, 173A3, and 176A3, were also Significantly afl‘ected for both initiation and elongation (Fig. 5). The 170A3 mutant was more defective in initiation than 173A3, but they had very similar defects in elongation. Perhaps more interestingly, the 176A3 mutant was partially active in initiation but inactive for elongation stimulation. The initiation assay is more complex than the elongation assay, because initiation is influenced by all of the general transcription factors and some regulatory factors in the extract system, and the elongation assay may only involve elongating pol H and TFHF. In the initiation assay, therefore, general or regulatory factors could partially complement TFHF activity. 17 6A3, therefore, might be partially complemented for its function in initiation by interaction with a general or regulatory transcription factor, which is present in the cell extract but absent in salt-washed elongation complexes. Conceivably, re-addition of such a factor to the elongation complex might relieve the 176A3 defect in elongation. Because the same region of RAP74 contributed strongly to both initiation and elongation, RAP74 may perform a Similar role in both processes. In preinitiation 100 complex assembly, the role of RAP74 appears to be to wrap DNA around pol H and to isomerize the complex (Robert et al., 1998). If RAP74 has a Similar role in elongation, it is also likely to involve DNA wrapping around pol H. In initiation, DNA wrapping is induced around eukaryotic RNA polymerase I (Schultz et al., 1993), RNA polymerase H (Kim et al., 1997; Robert et al., 1998) and prokaryotic RNA polymerase (Craig et al., 1995; Polyakov et al., 1995). It was somewhat surprising that the sequence requirements for RAP74 were so Similar for initiation and elongation, because a very different conclusion was reached for the RAP30 subunit of TFHF (Tan et al., 1995). Although RAP30 mutations that fail to bind RAP74 were found to be severely defective for both initiation and elongation, mutations in other regions of RAP30 afl‘ected initiation and elongation in different ways. RAP30 mutations within a presumed pol H binding region were defective in elongation stimulation but not in initiation. RAP30 mutations within a DNA-interacting region were defective for accurate initiation but not elongation stimulation (Tan et al., 1995). These results may indicate that RAP30 has distinct roles in initiation and elongation, although RAP74 appears to fulfill a common role in both processes. Because DNA-binding regions of RAP30 appear to be critical for initiation but dispensable for elongation, RAP30 may interact specifically with DNA in the preinitiation complex. During elongation the DNA sequence encountered by pol His in flux, and RAP30 may make less extensive template contacts. RAP30 is also likely to make reduced protein-protein contacts as initiation factors dissociate fiom the elongation complex. 101 Acknowledgements I thank Stephan Reimers, Drs. Delin Ren, Steven Triezenberg, Fan Shen, and Richard Burgess for proteins. Augen Pioszak, Victoria Sutton, Jessica Metcalf-Burton, Hiroe Taki, Julia Clay, and Nadine Kobty helped with production of RAP74 mutants. Dr. Ann Finkelstein was instrumental in setting up the gel mobility Shift assay. This work was supported by a grant from the American Cancer Society, by Michigan State University, and by the Michigan State University Agricultlu'al Experiment Station. Verna C. Finkelstein also contributed funds to support this work. 102 CHAPTER 3 SITE-DIRECTED MUTAGENESIS OF THE N-TERMINAL DOMAIN OF RAP74: CRITICAL AMINO ACID RESIDUES INVOLVED IN ELONGATION Introduction Accurate initiation from human pre-messenger RNA promoters requires the cooperation of RNA polymerase H (pol H) and general transcription factors (Orphanides .et al., 1996; Conaway and Conaway, 1993; Roeder, 1996). An ordered pathway for assembly of an active transcription complex on a promoter containing a TATA box has been defined in vitro (Orphanides et al., 1996; Conaway and Conaway, 1993; Zawel and Reinberg, 1993). TBP binds to the TATA element. TFHB then associates with TBP and promoter DNA to form a TBP-TFHB-promoter complex. TFHF escorts pol H into the TBP-TFHB-promoter complex, and TFIIF is required for stable binding and retention of . pol H. TFIIE further stabilizes the complex and is necessary to recruit TFIH-I. TFIH-I has ATP-dependent DNA helicase and CTD kinase activities. The TFIIH helicase activities are believed to open the DNA helix for formation of the open complex prior to initiation (Orphanides et al., 1996). Human TFIIF is an G252 heterotetramer of RAP30 and RAP74 subunits. Both subunits participate in recruitment of pol H to the promoter and both are necessary for accurate initiation fiom linear DNA templates in vitro (Lei etal., 1998; Tan et al., 1994). Deletion mutagenesis of the gene encoding the 517 amino acid (a) RAP74 subunit revealed an important region extending from as 136 to 217 (Lei et al., 1998; Chapter 2). RAP74(1-136) does not bind to RAP30 and is inactive in forming the transcription 103 complexes and in transcription assays. RAP74(1-172) forms transcription complexes readily but has weak activity in transcription. RAP74(1-205) is more active than RAP74(1-172), and RAP74(1-217) has Similar activity to RAP74(1-517, wt). TFHF was recently Shown to have a filndamental role in isomerization of the pol H preinitiation complex (Forget et al., 1997 ; Robert et al., 1998). Site-Specific photocrosslinking analyses of complexes containing an adenovirus major late promoter (AdMLP), TBP, TFIIB, TFHF, TFHE, and RNA polymerase H demonstrate that TFIIF induces promoter DNA to wrap tightly around RNA polymerase H and the general factors. TFIIF containing RAP74(1-172), although fully assembled, did not support the tightly wrapped DNA structure. TFIIF containing RAP74(1-205) and more complete versions of RAP74, however, did support this isomerized and more active structure. The region of RAP74 between aa 172 to 205, therefore, appears to have a critical function in complex isomerization, and the isomerization appears to involve tight DNA wrapping around pol H and the general factors. In addition to its roles in preinitiation complex assembly, isomerization and initiation, TFHF also stimulates the rate of elongation by RNA polymerase H (Price et al., 1989; Izban and Luse, 1992a; Bengal et al., 1991). Both the RAP30 and RAP74 subunits are required for elongation rate stimulation (Lei et al., 1998; Tan et al., 1994). Interestingly, RAP74(1-136), (1-172), (1-205), (1-217) and (1-517, wt) were found to have very similar activities in both initiation and elongation stimulation, indicating that the region of RAP74 between a 136 to 217 makes a similar contribution to multiple stages of the transcription cycle (Lei et al., 1998). Because DNA wrapping induced by TFHF has been Shown to be important for initiation, perhaps DNA wrapping around 104 RNA polymerase H is a feature of rapid elongation complexes as well. The region of RAP74 between a 136 and 217 is highly conserved from yeast to human. In this chapter we report a detailed mutational analysis of the RAP74 subunit of TFHF in this region. 105 Materials and Methods Construction of RAP74 mutants Site-directed mutants of human RAP74 were constructed using the Quick Change Site-Directed Mutagenesis Kit (Stratagene) and appropriate primers. Mutations were confirmed by DNA sequencing. RAP74 mutant proteins were produced in E. coli, pmified to near homogeneity, and assembled with recombinant human RAP30 to generate TFHF complexes (Wang et al., 1993; Wang et al., 1994). Transcription assays Immobilized templates Immobilized templates were prepared as described (Lei et al., 1998; Chapter 2). The template for amplification and the upstream biotinylated primer were the same as described (Chapter 2). The sequence of the downstream primer was 5'- GTGCT‘CATCAT'I‘GGAAAACGTTCTT-3'. The template extended fiom positions -263 to +1 ,250 relative to the transcription start (+1). Biotinylated DNA was incubated with streptavidin paramagnetic beads (Vector Laboratories) in 2 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7 .5 for 15 min at room temperature. Immobilized templates were collected with a magnetic particle separator (CPG), washed 2 times and stored at 4°C in phosphate buffered saline pH 7.2 containing 1 mg/ml BSA and 0.03% NaN 3. Initiation assay: pulse-sarko_syl chase This was modified hour the pulse-chase protocol previously described (Chapter 2). 0.25% sarkosyl was added during the “chase” to prevent premature termination. Each 20 ul reaction mixture contained 800 ng DNA template (pML digested with Sma I at +217), TFHF-depleted extract (72 ug total protein), recombinant TFHF or TFIIF 106 mutant (10 pmol each), in transcription buffer (12 mM HEPES, pH 7.4, 12% glycerol, 0.12 mM EDTA, 0.12 mM EGTA, 1.2 mM DTT) containing 60 mM KCl and 12 mM MgC12. Preinitiation complexes were formed for 60 min at 30 0C. 100 uM ATP, CTP, GTP and 1 uM a32P-UTP (10 uCi per reaction) were added to initiate transcription for l min. 1 mM ATP, UTP, GTP, CTP, and 0.25% sarkosyl were added, and elongation continued for 30 min. Reactions were stopped by addition of 200 111 0.1 M sodium acetate (pH 5.4), 0.5 % sodium dodecyl sulfate, 2 mM EDTA, and 100 ug/ml tRNA, followed by phenol-chloroform extraction and ethanol precipitation. Samples were electrophoresed in a 6% polyacrylamide gel containing 50 % urea (w/v). For quantitation of the gel, the Signal in the presence of RAP30 and the absence of added RAP74 was used to estimate background (Figure 1, lane 11). The weak Signal obtained in the absence of added RAP74 was attributed to residual RAP74 remaining in the TFIIF- depleted extract. Elon ation stimulation assa : sark l and salt wash This was modified fi‘om the method previously described (Chapter 2). A 20 ul reaction mixture consisted of 2 ul paramagnetic beads carrying the adenovirus major late promoter, transcription extract derived fi'orn HeLa cell nuclei (108 ug total protein), and transcription buffer containing 60 mM KCl and 12 mM MgC12. Samples were incubated 60 min at 30 0C to form preinitiation complexes. Transcription was initiated by addition of 100 uM ATP, GTP, CTP, and 1 uM (10 uCi) a—32P-UTP (2 ul volume). After 1 min, elongation complexes were washed extensively with transcription buffer containing 1% sarkosyl, 500 mM KCl and 1 mg/ml BSA. This treatment appeared to be effective in removing the accessory factors because the elongation rate of pol H was strongly 107 stimulated by TFHF (Figs. 1, 3, and 4). Complexes were then washed with transcription buffer containing 60 mM KCl and 1 mg/ml BSA. Complexes were resuspended in 20 ul transcription buffer containing 60 mM KCl and 12 mM MgC12, recombinant TFHF complexes were added (20 pmol each or as indicated) and incubated for 5 min. 1 mM ATP, GTP, CTP, and UTP were added in 2 ul, and transcripts were elongated for various amounts of time as indicated. Transcripts were isolated by phenol extraction and ethanol precipitation and electrophoresed in a 10% polyacrylamide gel. The gel was quantitated using a Phosphorimager (Molecular Dynamics). 108 Results Detailed mutational analysis of RAP74 in the critical rggion between as 136 and 217 Previous work had indicated that the N-terminal domain of RAP74 was very important for both initiation and elongation of transcription (Lei et al., 1998; Chapter 2). This hypothesis was tested in the experiment shown in Fig. 1, using human recombinant TFHF containing RAP30 and RAP74 or a RAP74 deletion mutant. For these studies, improvements were made in accurate initiation and elongation assays. For the initiation assay, an extract derived fi'om human HeLa cells was depleted of TFHF by immunoprecipitation with anti-RAP30 and anti-RAP74 antibodies. Recombinant TFIIF or a TFIIF mutant was added back to the depleted extract to restore accurate initiation. The template for transcription was the plasmid pML containing the adenovirus major late promoter. The DNA was treated with restriction enzyme Sma I that has a cleavage site at position +217 relative to the transcription start Site. Transcription was initiated with a l min pulse of 100 M ATP, CTP, GTP, and 1 uM a32P-UTP. Sarkosyl was then added to 0.25 % and elongation continued for 30 min with 1 mM each NTP. In this protocol, sarkosyl removes elongation and termination factors from RNA polymerase 11 so that initiated chains are recovered efficiently at the +217 position (data not shown). As a result, this runofl‘ assay very accurately reflects Single-round initiation fi'om the adenovirus major late promoter. Elongation complexes were prepared on bead templates containing the adenovirus major late promoter. In an extract derived from HeLa cell nuclei, transcription was accmately initiated fi'om the promoter with a l min pulse of 100 11M ATP, CTP, GTP, and 1 11M a32P-UTP. Nascent elongation complexes were washed free of elongation and 109 Figure 1. Identification of a region of RAP74 that is critical for transcription initiation and elongation stimulation. A) Accurate initiation from the adenovirus major late promoter. An extract derived from HeLa cell nuclei was depleted of TFIIF by immunoprecipitation with anti-RAP30 and anti-RAP74 antibodies. Activity was restored by addition of 10 pmol TFIIF formed with RAP74 (1-517, wt) (designated F517) or deletion mutants F217, F205, F172, or F136. The reaction in lane 12 was identical to those in lanes 1 and 2 except that 1 rig/ml a-amanitin was included in the reaction. B) Phosphorimager quantitation of the data shown in “A”. C) Elongation stimulation by TFIIF deletion mutants. Elongation complexes were formed on immobilized templates close to the adenovirus major late promoter and washed with 1% sarkosyl and 0.5 M KCl to remove nascent elongation factors. 20 pmol TFIIF was added to washed elongation complexes and 1 mM each NTP was added for 30 3 (samples designated “chase”). M is a single-stranded, 5’ end-labeled DNA marker. llO OE 9915 ZLId SOZJ LIZJ L195 runoff-> OOOO... 4e 23456789101112 1 m + ,_ .i M? + 0‘ ~ ~51 en + 951:1 ‘ (a. + 90“ q a + lerl : ‘. + 119d ‘ Q) LI-t P in . I \ \ s .3 § §§§§§ § § e 8 3 a: 7, U 53 120 111 100 — l l 1 O O O OO \O V- (%) uoyldlrosunll F217 F205 F172 F136 30 F517 ten 1'84 858 (Fit 00D pre. the. flip bot‘ imp Drc PA termination factors with buffer containing 1% sarkosyl and 0.5 M KCl. Complexes were re-equilibrated with transcription buffer, and TFHF was added to the reaction. For the assays Shown in Fig. l, elongation was for 30 seconds in the presence of 1 mM each NTP (Fig. 1C). TFHF containing RAP74(1-217) had almost as much activity as wild type TFHF in initiation and Slightly higher activity than wild type TFHF in elongation. TF HF containing RAP74(1-205), which is minimally required to support isomerization of the preinitiation complex, had reduced activity in both assays. RAP74(1-172) was further reduced in activity, and RAP74(1-136) was inactive. RAP74(1-136) is the only one of these mutants that does not stably assemble with the RAP30 subunit of TFHF. This experiment Shows that amino acids 136 to 217 of human RAP74 are very important for both accurate initiation and elongation of transcription. To determine which amino acids in the region fi'om aa 136 to 217 were most important for RAP74 firnction, a large set of Site-directed mutants was constructed (Fig. 2). In Fig. 2A an alignment of human RAP74 with homologues fi'om Xenopus, Drosophila, and yeast is shown. Pl-H) analysis indicated that aa 157 to 182 of human RAP74 may constitute an extended or-helix and this structure is likely to be preserved in RAP74 homologues. Single, double, and triple amino acid substitutions are indicated beneath the sequence and categorized according to their activities in initiation and elongation assays (Figs. 3-6). Single substitutions are named. according to the RAP74 amino acid that is substituted, L155A, W163A, etc. Multiple mutants are named for the 112 Figure 2. TFIIF mutagenesis. A) RAP74 homologues from eukaryotic Species including human (h), Xenopus (x), Drosophila (d for Drosophila Factor 5a), and S. cerevisiae (y for yeast Ssu7 l , suppressor of sua 7; also designated ng1 for transcription factor “g”). PHD analysis indicates that a large segment of this sequence is (Jr-helical (hhhhhhh). B) RAP74 mutants analyzed in this study. Mutants are designated as “Critical”, “Moderate”, or “Less important” depending on their observed defects in initiation and elongation (Figs. 3-6). The most important residues are boxed in the sequence. Underlined mutants have slightly different properties in initiation and elongation assays (Fig. 6). 113 N enema 44 444 444 mmm xxx 4 4 «14 m 4 4 4 4| 4 444 444 mmrm xxx 4 4 4 4 4 4 mmm 4 4 4 4 4 ncmuaoeefl mmoq m m l l 444 444 4 444 4 4 4 ononmpoz 4 4 444 4 Hmoflumuo SE 444 4 mrmm xxx xx 4 SN mzmqoomqomqummfimmommmmmmmomooaooxqmmogmmggmmfigfiafifimegmm4mm4wcmu 2.! 434m: oam oom oma oma 0:” 03” 0.3 ova QMH _ smarts m m gang; engage . m2. causeway“. mmwmmzoami :oomefiooogfi SEER ........ EmegecazaamgmmfiemgmxgeH§e4>u2m4§§x§enm§m mam a $4 gqaamqofixanmmgwxem ....... 9334. 4mmmedmmmmnem§n§mm§ ..... fiemmomfifimsmmwmoumomgeamsfizeofi S; m emm wfiacamacmaxqammxmxmmxou ........ commaxooommamamneeooxqmmoofigram—332 ..... ammzmoflmflsqemggagmaafiea and x Sm msmgccmaofimumgmmemxmx .................. wwwomaogmmogm . , . . . .. s at... a o2 n cam oom omH omH ova omH omH ova OMH 114 position of the first substituted amino acid, 161K3, 176A3, etc. A combination of alanine substitutions and charge-reversal mutations was constructed based on the notion that these mutations would cause changes in activity without inducing long-range changes in protein conformation. Human recombinant RAP74 mutants were produced in E. coli, purified to near homogeneity, combined in vitro with human recombinant RAP30 in buffer containing 4 M urea. The TFIIF complex was reconstituted by dialysis into buffer without mea (Wang et al., 1994). Mutants with the most pronounced transcriptional defects have been analyzed by gel filtration chromatography. This analysis indicates that RAP74 mutants form 01202 heterotetramers in complex with RAP30 as does wild type TFHF (Wang et al., 1994; data not shown). TFIIF mutants affect both initiation and elongation vegy similaru Mutants were analyzed for their ability to stimulate the elongation rate of RNA polymerase H (Fig. 3). As described in Fig. 1, sarkosyl- and salt-washed elongation complexes were prepared, a TFHF sample was added, and elongation was continued with 1 mM ATP, GTP, CTP, and UTP for 15, 30, or 60 S. A phosphorimager was used to determine the average length of transcripts in each gel lane and this value was plotted versus the elongation time. The Slope of the line is reported as the average elongation rate. Quantitation of elongation rates was very precise in single experiments with an r2 of 0.99 to 1.0. Variation between experiments for a TFHF sample was less than 10 % (data not Shown). The value for TFHF wt was used to scale data between experiments. RNA polymerase H pauses at many Sites along the template. The selection of pause Sites and the efficiency of pausing appear to be identical whether TFIIF is present or absent fiom 115 Figure 3. Analysis of representative TFIIF mutants in elongation. A) The elongation rate stimulation assay. Washed elongation complexes were prepared as described in Fig. 1. After addition of 20 pmol TFIIF, 1 mM of each NTP was added for 15, 30, or 60 S as indicated (chase). The size marker (M) is a 5’ end-labeled, Single-stranded DNA marker for estimation of transcript sizes. TFIIF mutants are named for the RAP74 mutations they contain (i.e. L171A). B) Quantitation of the data shown in “A”. A phosphorimager was used to estimate the midpoint of the distribution of RNA bands. Average transcript length is plotted against elongation time. The Slope is reported as the average elongation rate. Slopes vary between 1'2 of 0.99 and 1.0. 116 11F none 1-517 1-217 L171A F138A V170A W164A 1176A 1841(3 1-136 chase -/I/|/lé]/l/1/lélélél 587—. 434—11) | | ‘ 267—4- ' .' .d 213—: .a q " - 184 -" r. 1.- ‘~ .a h- is.» I- ‘ ‘ 124—0 .""T.".'“ h. g‘ i:- 104-0 t- . 1111:. 89 -. ,H 80 -: a" , 3 3, i .- r 64 -- .1 . 57 — -7 T 8 f : '.. .. e 51 —-.. I“ 3 2' ’ cocoooggogocOQ .- -.. " ' ‘ nee—4‘ _”:' M135791113151719212325272931 24681012141618202224262830 700 +l-Sl7 600,-0—1-217 +Ll7lA —r&-Fl38A 50° +v170A c +184K3 5 . E400 ~I—wr64A g -E1-1176A 3004—1436 (— ~0-none 200~ 1004 o// 0 I T T M l l o 10 20 30 4o 50 60 7o Time(seeond) Figure3 117 Figure 4. TFIIF containing RAP74(1176A) has a similar affinity as TFIIF wt for elongation complexes but a lower activity. A) Elongation stimulation by TFIIF wt and TFIIF containing RAP74(1176A). The chase was for 30 s with 1 mM each NTP. Lanes 2-7 and 8-12 contained 5, 10, 20, 40, or 60 pmol of the indicated TFIIF complexes. B) Quantitation of the data shown in “A”. A phosphorimager was used to estimate the average Size of the RNA transcripts in each lane. 118 a chasm cone "Ema 8 on ow on ON 2 o <05 _ _ 14. w :2 i . J . cm 8— em— .08 omm 8m (in) srdrrosuarl aflmAv m Ion IS I. o: I 3. II b: I 8. l NVN 1. cNm 11 3V II 8n <3: 119 the reaction (Fig. 3 and data not Shown). TFHF appears to decrease the dwell time at all Sites. TFHF containing RAP74 mutants within this N-terminal domain appeared to have a very Similar affinity for transcription complexes compared to TFHF containing RAP74 wt (Fig. 4, and data not shown). Several lines of evidence supported this claim. First, the ability of TF HF mutants to form the DNA-TBP-TFIIB-pol H-TFHF complex was tested by a gel mobility shift assay (Lei et al., 1998; Chapter 2). Even the most defective TF HF mutants, except 1-136 which is completely inactive, recruited pol H as efficiently as TFHF wt (unpublished results, Delin Ren and Zachary Burton). Second, TFIIF mutants competed with TFHF wt in initiation assays (Lei et al., 1998; unpublished results, Delin Ren and Zachary Bmton). Addition of a lO-fold molar excess of a TFIIF mutant (such as 117 6A) over TFHF wt reduces transcription to the level supported by the mutant alone. Furthermore, the concentration of TFHF necessary to support accurate initiation and elongation stimulation was determined. There was no clear difference between the concentration requirement for TFIIF wt and TFIIF mutants either for accurate initiation (unpublished results, Delin Ren and Zachary Burton) or for elongation stimulation (Fig. 4). Additionally, these experiments showed that the concentrations used for analysis of mutants were saturating for both initiation and elongation (Figs. 1, 3, and 6), so low activities of mutants were due to Specific defects other than an altered affinity for transcription complexes. . The elongation rates of TFHF mutants are compared in Fig. 5. TFIIF containing RAP74(1-136) is inactive for elongation stimulation. RAP74(1-158), however, and all substitution mutants within the as 136 to 217 region have some ability to stimulate 120 Figure 5. The average elongation rates of TFIIF and TFIIF mutants. Elongation stimulation assays were done as in Fig. 3. An asterisk (*) indicates that a TFIIF mutant was constructed in RAP74(1-217) rather than RAP74(1-517, wt). 121 1-517 1-217 [-205 1-172 r-rss 1436 None r13“ P139A V140A WI43A YIMA NIASA F138AIN145A F14“ L149A 15183 1154A LISSA TISSA 158K: E15“ ElflA [6116' £16“ Elm £163A WIMA 111654 16623' 3166A to‘MJ' 3161A mm K169A AUTO-177) l70A3' V170A' Ll7lA' Nlm' mm 173A3‘ 8173A “74A 8175A 176A3' 1176A M177A le’ 179A3' le' 18410“ 1881(3' 191 KT 19433' 199!!! 204A? 207A? 210A3’ 213A? Elongation Rate (nt/min) -- N u «b Us ON \1 8 8 8 8 8 8 8 500 528 292 178 154 92 94 516 545 502 480 500 548 586 499 543 520 327 173 302 204 452 298 182 475 470 468 177 504 154 489 239 249 276 470 225 176 260 524 186 179 217 239 372 584 163 149 171 532 244 496 204 399 304 530 555 484 502 495 497 Figure 5 122 elongation. Substitution mutants having the lowest elongation rates (less than 200 nt/min) include L155A, 161K3*, W164A, 166E3*, 170A3*, N172A*, N172A, 176A3*, 1176A, and M177A. L155, W164, N172, 1176, and M177 all have hydrophobic Side chains that appear to be critical for RAP74 flmction. It is likely that these amino acid residues form a hydrophobic patch important for protein-protein interactions. Single alanine substitutions also indicate that the individual side chain beyond B-carbon at the positions ofaa 161 (E), 162 (E), 163 (E), 166 (R), 167 (R), 169 (K), 170 (V), 171 (L), or 178 (Q) is not critical for RAP74 function. The defects associated with 161K3* and 166E3“ are caused by the combinations of multiple substitutions, because none of the Single substitutions at these positions is critical. The C-terminal domain and the central region do not afl‘ect elongation because the same substitutions constructed in either RAP74(1-517, wt) or RAP74(1-217) have Similar effects (compare N172A and N172A*, and data not shown). Interestingly, several substitutions, such as P139A, N145A, F138A/N 145A, L149A, Sl75A, and 199E3* appear to increase the elongation activity of RAP74. Initiation and elongation activities for the entire collection of TFIIF mutants are shown in Fig. 6. The striking result from this analysis iS that mutants that are adversely affected for elongation rate stimulation are similarly affected for accurate initiation. AS previously Shown, TFHF containing RAP74(1-136) is inactive for accurate transcription and elongation stimulation (Fig. 1). RAP74(1-158), however, and all substitution mutants within the a 136 to 217 region have some ability to support accurate initiation and elongation stimulation. As the data is displayed, the elongation rate data appear to mirror the results from the initiation assays, demonstrating that amino acids in this region 123 of RAP74 make very similar contributions to both the initiation and elongation phases of the transcription cycle. 124 Figure 6. TFIIF mutants have very Similar activities in accurate initiation and elongation rate stimulation. The activities of TFIIF mutants in elongation were compared with their activities in initiation. Accurate initiation assays were done as in Fig. 1. 125 \l ,8 Elongation Rate (at/min) Initiation (°/o) 8 ‘8 8 ‘8 8’ '8‘ o 8 e a e 8‘ s a 119 4" 100 8Z9 4172 Z62 44 8L1 —- l9 9S1 —- l8 Z6 0 V6 O 915 4—475 svs J—i 68 LLI — 23 1705 J". 93 791 — 22 68V 41 60 672 l 65 9LZ J—i 63 0L? 9 88 SZZ — 23 9L1 — 21 092 —— 39 7Z9 —— 50 981 _ 33 6L1 —— 33 682 —; A 69 ZL‘E — #l-i 68 V89 gi-l 110 C91 19 6H 17 11.1 21 285 #l—i 62 WZ ——" 56 9617 F4 86 WZ J 58 668 J 63 1708 l 78 069 —=: Jr 73 999 J-—i 65 E 1. Z09 ’4 65 56? ‘ '—| 70 L617 — 3 73 126 Discussion The region of RAP74 between a 136 and 217 is very important for accurate initiation fi'om the adenovirus major late promoter and elongation rate stimulation. It is striking that RAP74 mutants in this region have almost equivalent effects in such different assays. The accruate initiation assay was done in a reconstituted HeLa cell extract system. Transcription from the adenovirus major late promoter was dependent on: 1) ATP hydrolysis; 2) presumably, all of the general transcription factors; and 3) the presence of the DNA binding Site for the transcriptional activator USF/MLTF (upstream sequence factor/maj or late transcription factor). Many general and regulator proteins, therefore, could modulate TFHF activity in this system. Because of extensive washing of elongation complexes, the elongation stimulation assay is likely dependent only on RNA polymerase H that iS transcriptionally engaged and TFHF. It is therefore a surprise that results in the initiation and elongation assays mirror one another so closely. It is possible that the TFHF activity in these assays depends solely on interactions between TFIIF and RNA polymerase H and/or the DNA template. The L155 to M177 region of RAP74 is conserved in evolution and is likely to form an a—helical structure (Fig. 2). Amino acid substitutions in L155, W164, N172, 1176 and M17 7 have the greatest defects in accruate initiation and elongation. A common feature of these amino acid residues is that they all have hydrophobic Side chains that are likely involved in protein-protein interactions. These mutants, however, appear by various criteria to enter transcriptiOn complexes with wild type affinity (Fig. 4 and data not Shown). Unfortunately, the molecular structure of this region of RAP74 has not yet been reported. Mutagenic analysis indicates that, if this region is (ll-helical, there 127 is no particular relationship between the face of the helix on which Side chains are displayed and the transcriptional defects of substitutions at those positions. In the future, a molecular structure of human TFHF wiH assist the interpretation of these mutants. The region between as 172 and 205 is important for self-association of RAP74 in protein affinity chromatography experiments (Robert et al., 1998). The critical mutations identified in this work, therefore, may lie within a dimerization region that is important in maintaining the TFIIF heterotetramer. The gel filtration chromatography analysis of the native molecular weight of TFHF samples, however, indicated that even the most severely affected mutants in this region, including RAP74(Il 7 6A), RAP74A(170-1 7 7 ) and RAP74(1-1 5 8), formed heterotetramers with RAP30 (data not shown). Site-specific DNA-protein photocrosslinking studies have recently demonstrated that the region of RAP74 between a 172 and 205 is critical for forming a tight wrap of adenovirus major late promoter DNA around a preinitiation complex containing TBP, TFHB, RNA polymerase II, TFHF and TFHE (Robert et al., 1998). RAP74(1-172) assembles into this complex but fails to support a tightly wrapped structure and fails to support formation of many Specific photocrosslinks to general transcription factors and RNA polymerase H within the core promoter. RAP74(1-205), on the other hand, appears to support DNA wrapping, apparently with the same efficiency as RAP74 wt. RAP74(1- 205) is not as active in transcription as RAP74 wt (Fig. l), but its transcriptional defect has not yet been revealed in photocrosslinldng experiments. The interpretation of the photocrosslinking data was that the region of RAP74 between aa 172 and 205 is necessary for forming the tight DNA wrap around RNA polymerase H and for isomerization of the preinitiation complex. Isomerization was hypothesized to include 128 tight wrapping of promoter DNA around pol H and the general factors and Sharp bending of DNA through the RNA polymerase H active site. The critical region between L155 and M177 that we identified by mutational analysis appears to correspond to this region of RAP74 that is critical for isomerization. Dr. Benoit Coulombe’s laboratory is currently testing these amino acid substitution mutants in photocrosslinking experiments to determine whether they can support a tight promoter DNA wrap around pol H. Assuming that the defect of these RAP74 mutants in initiation is due to a failure to isomerize the preinitiation complex, what iS their defect in elongation? Because these mutants have such Similar effects on initiation and elongation, they may also fail to properly isomerize the elongation complex. Because DNA wrapping and bending appear to be the primary characteristics of the isomerized preinitiation complex, we hypothesize that Sharp DNA bending through the RNA polymerase active Site may Similarly stimulate elongation by RNA polymerase H and that TFIIF supports this bent structure during elongation. A model to describe these ideas is Shown in Fig. 7. This model takes into consideration: 1) asynchrony of RNA polymerase II elongation; 2) DNA bending and possibly wrapping in the elongation complex; 3) studies of stalled elongation complexes; 4) isomerization of the elongation complex induced by TFHF; and 5) the observation that pause Site selection is not affected by TFHF. RNA polymerase H elongates transcription asynchronously, as if polymerase iS partitioning between active (EC*) and inactive (EC) forms during elongation. Asynchronous elongatiOn has been described most clearly for RNA polymerase IH, although this also is 129 Figure 7. A model for the role of TFIIF in isomerization of the elongation complex. TFIIF is proposed to affect partitioning between unisomerized and isomerized complexes during elongation. TFIIS is suggested to work on the unisomerized elongation complex to overcome irreversible transcriptional arrest. Isomerization of complexes is suggested to involve wrapping of template DNA around RNA polymerase II and Sharp bending of the template through the RNA polymerase active site. 130 xanoo 09.08202 umwtm Wm“:a.seamennestmnxvfl“mtuawmxwwrmmu..1. .1 . .11....3'. NZIACVOM a. cogs 9:8 m __ .8 o5 8:25 42o been 2 uses m 4.38358 :80 834:8 m s as“: as been. 26. __m\m_E “ls/NZ Mr Ed 88.. cozmmcofi 526.4. _n_n_ .. m 0m 8.35 a 8.58 558 r Eom 3+ch 3+ch A~+5nez fenez sum AHV 0m 8:85an 3+5; are: 2+5: 2+5; A53. E: asses as“: _ strum 2.15.3 from 31.5.». 3145.3. 3+5 .5 an A NEE? _nn :53. nonsense 8...“: e0.x. 853.com. 2: 83:88.. “as: .8 been coca: zcvacrv: .6139. cease—3.8 .38“— ..om caucuses. Figure 7 131 a feature of RNA polymerase II and E. coli RNA polymerase (Matsuzaki et al., 1994). In this model, EC* is depicted with a DNA template that is sharply bent through the RNA polymerase 11 active site, and the template is less sharply bent in EC. Our interpretation of studies of stalled elongation complexes is that the stalled complex must resemble the unisomerized EC, which is more likely to be an entry point into an editing mode than an active elongation mode. Stalled elongation complexes do not appear to interact strongly with TFHF (data not shown), although TFIIF stimulates elongation about 6-fold (Figs. 3 and 6). Stalled elongation complexes have short exonuclease III, DNase I, and hydroxyl radical footprints, indicating that the DNA template is not wrapped armmd RNA polymerase in EC (Samkurashvili and Luse, 1998; Selby et al., 1997). TFIIS/SII may interact with the EC form to resolve backtracked complexes (Izban and Luse, 1992b; Izban and Luse, 1993a; Awrey et al., 1998). The proposed role of TFIIF in this model is to help support the EC* form of the elongation complex. TFIIF might do this by accelerating k; and inhibiting k5. TFIIF is proposed to help maintain the DNA template in a sharply bent conformation through the RNA polymerase II active site during elongation. TFHF is not expected to interact directly with the RNA polymerase II active site. In this regard, TFIIF may not affect k*(n), the rate of phosphodiester bond formation. If TFIIF affected this step, TFIIF would alter the selection of pause sites, which has not been observed. 132 Acknowledgements I thank Dr. Delin Ren for all the RAP74 substitution mutants used in this study. I also thank Dr. Delin Ren for sharing the unpublished results on the analysis of RAP74 mutants in the gel mobility shift assay and initiation assay. This work was supported by a grant from the American Cancer Society, by Michigan State University, and by the Michigan State University Agricultural Experiment Station. 133 CHAPTER 4 FUNCTIONS OF THE C-TERMINAL DOMAIN AND CENTRAL REGION OF RAP74 IN RECYCLING OF RNA POLYMERASE II INTRODUCTION An intriguing feature of the RPBl subunit of pol II is the carboxy terminal domain (CTD) which has the consensus sequence YSPTSPS tandemly repeated 52 times in human pol II (Dahmus, 1996). Phosphorylation and dephosphorylation of the CTD by the regulated activities of CTD kinases (F eaver et al., 1991; Lu et al., 1992; Marshall et al., 1996; Serizawa et al., 1992; Serizawa et al., 1993) and phosphatases (Chambers and Dahmus, 1994) appear to control progression through the transcription cycle. Pol II enters the preinitiation complex with its CI'D in a largely unmodified form designated pol IIA (Laybourn and Dahmus, 1989; Lu et al., 1991). During elongation, pol II is converted to the pol no form (Bartholomew et al., 1986; Laybourn and Dahmus, 1989) which is heavily phosphorylated on the SP serines of the YSPTSPS consensus sequence, so hyperphosphorylation of the CTD is thought to be important to establish and maintain the elongation complex. Removal of phosphates from the CTD may be a signal to terminate transcription and recycle pol II to a promoter. A recently identified CTD phosphatase that catalyzes the dephosphorylation of pol 110 to pol HA is stimulated by the C-terminal domain of the RAP74 subunit of TFIIF (Chambers et al., 1995). Interestingly, RAP74-dependent stimulation of CTD phosphatase activity is blocked by addition of TFIIB. The C-terminal domain of RAP74 binds to TFIIB (Fang and Burton, 1996) and pol H (Wang and Burton, 1995), so TFIIF, TFHB, and the CTD phosphatase may be components of a multiprotein complex that 134 binds pol H and regulates pol II recycling. In this chapter we demonstrate that the central region and the C-terminal domain of RAP74 stimulate multiple-round transcription in an extract system consistent with a role- for RAP74 in transcriptional recycling. 135 Materials and Methods Transcription factors and extracts Recombinant human RAP30, RAP74, and RAP74 mutants were prepared and quantitated as described (Chapter 2). Construction of new RAP74 mutants is described below. The TFIIF-depleted extract was completely dependent on the re-addition of RAP30 for activity and was strongly stimulated by addition of RAP74. Construction of RAP74 mutants Internal deletion mutants RAP74(A306-351), (A276-351), and (A219-351) were constructed using the Quick Change Site-Directed Mutagenesis Kit (Stratagene). All mutants were made using the same primer for the amino acid 351 position, 5'- GACATTGACAGCGAGGCCTCCI‘CAGCCCTCTI‘CATGGCG-3'. For the second oligonucleotide primers, 5'- GGAGGCCI‘CGCTGTCAATGTCGCT CT GCT CATCGACACCATI‘GGG-B', 5'- GGAGGCCI‘CGCI‘GTCAATGTCTGACATGTAGTCCACCI‘C'ITGGCC-B', and 5'- GGAGGCCT C GCTGTCAATGTCGGAGGACA’ITI‘CCAGGTCGTCTTCAAGGTC-3' were used, respectively. The underlined sequences represent the complimentary overlap between the two mutant primers. Mutated RAP74 genes were confirmed by DNA sequencing. Multiple-round and sarkosyl block assays Transcription was initiated fi'om an adenovirus major late promoter template digested with Smal to produce a +217 base runoff transcript. The source of transcription factors was TFIIF-depleted extract (7 2 ug total protein) supplemented with recombinant RAP30 and RAP74 (10 pmol each, except as noted). For all reactions, preinitiation 136 complexes were formed for 60 min at 30 0C. For the multiple-round assay, 600 uM ATP, CTP, GTP and 25 uM a32P-UTP (10 uCi per reaction) were added and transcription continued for the indicated times (Figs. 1-3). For the 10 min cold-multiple rormd assay, 600 uM ATP, CI‘P, GTP and 25 uM UTP were added and incubated for 10 min, and then a32P-UTP (10 uCi per reaction) was added and transcription continued for 60 min (Fig. 1). For the single-round sarkosyl block assay (Hawley and Roeder, 1985), 600 uM ATP, CTP, and 25 uM a32P-UTP (10 uCi per reaction) were added and incubated for l min. 600 uM GTP and 0.25 % w/v sarkosyl were added and transcription continued for 59 min (Fig. l), 79 min (Fig. 2B and 2C), or 99 min (Fig. 3A). 0.05 % sarkosyl was previously shown to be sufficient to block new initiation by pol II (Hawley and Roeder, 1985), so sarkosyl was added in five-fold excess over the amount necessary to constrain transcription to a single round. As a control, sarkosyl was added to reactions before NTPS, causing initiation to be completely blocked, indicating that the level of detergent added in experiments was sufficient to block reinitiation (data not shown). For the experiment shown in Fig. 2A, using the multiple-round transcription protocol, 0.25 % sarkosyl was added at the indicated times to block new initiation, and transcription was continued for an additional 30 min to complete all previously initiated chains (Hawley and Roeder, 1985). This was a control experiment to demonstrate that new initiation occurs throughout the course of the reaction. Transcription was quantitated and background selected as described above. G-less cassette ml 11 trap To characterize multiple-round transcription in the extract system, a "G-less cassette” trap for pol II was used (Szentirmay and Sawadogo, 1994). The template was 137 plasmid pML(C2AT)l9, the kind gift of Michele Sawadogo, containing the adenovirus major late promoter fused to a G-less cassette at position +11 (Sawadogo and Roeder, 1985a; Sawadogo and Roeder, 1985b). Preinitiation complexes were formed for 1 hr. Reactions contained TFIIF-depleted extract supplemented with 10 pmol recombinant RAP30 and RAP74. 600 uM ATP, GTP, and CTP, 1 mM 3'-O-methyl GTP, and 25 uM o32P-UTP (10 uCi per reaction) were added to reactions as indicated in Fig. 4. At t = +1 min, 0.05 % sarkosyl was added to some reactions to estimate single-round transcription. In the presence of ATP, CTP, UTP, and 3'-O-methyl-GTP a transcript of 390 bases was synthesized. Under this condition, pol II stalled after insertion of 3'-O-methyl-GMP into the RNA chain at position +390. The template was digested with Pqu to allow simultaneous detection of stalled transcription at +390 and runofl' transcription at position +602. In control experiments 0.05 % sarkosyl was found to be suficient to constrain transcription to a single round (data not shown). 0.05 % sarkosyl was used in this experiment because, unexpectedly, the early elongation complex formed from the G-less cassette template was much more sensitive to disruption with sarkosyl than that initiated from the wild type promoter (data not shown). The promoters in these two plasmids are identical from positions -256 to +10, and therefore it appears that the DNA sequence downstream fi'om +10 may contribute to the observed difference in sarkosyl sensitivity. For chase reactions, 1 mM GTP and UTP were added and elongation continued for 10 or 60 min, as indicated in Fig. 4. 138 RESULTS The central rggion and C-terminal domain of RAP74 stimulate multiple-round transcription RAP7 4 mutants were tested for the ability to support multiple-round transcription in vitro (Fig. 1). In one protocol, NTPs were added with a32P-UTP radiolabel and transcription was allowed to continue for 60 min. In a modified protocol, unlabeled NTPs were added for 10 min before addition of radiolabel (10 min cold—multiple round), and transcription continued for 60 min. For comparison, a single-round protocol Was done in which sarkosyl (0.25 %) was added 1 min after addition of NTPs to block new initiation (Hawley and Roeder, 1985). The final specific activity of radiolabel was the same in all three procedures, so the intensities of transcription signals can be compared directly. For the sarkosyl block assay (Fig. 1A), the observed transcriptional activities of RAP74 mutants were very similar to those determined in the pulse-spin initiation assay (Chapter 2). . Cycles of transcription were estimated by dividing the yield of transcripts in the absence of sarkosyl (mulfiple-rormd) by the yield of transcripts in the presence of sarkosyl (single-round). By this estimation, full length RAP74 supported approximately four cycles of transcription in 60 min (Fig. 1B). However, RAP74(1-409), (1-356), (1- 296), and (1-217) supported only about two rounds of accurate transcription. The region between a 409-517, therefore, was important for multiple round transcription. For RAP74(1-517), (1-409), and (1-356), cycles of transcription were not diminished using the 10 min cold—multiple round protocol. The transcription system, therefore, did not become limiting for factors or substrates within 70 min. In contrast, RAP74(1-217) and 139 Figure 1. The C-terminal domain of RAP74 stimulates multiple-round transcription. A) Single-round and multiple-round transcription assays. All samples contained TFIIF- depleted extract (DE; 72 pg protein), recombinant human RAP30 (10 pmol) and RAP74 or a RAP74 mutant (10 pmol). In the single-round protocol, reinitiation was blocked by addition of the anionic detergent sarkosyl. In the multiple-round protocol, transcription was allowed to proceed for 60 min. In the 10 min cold--multiple round protocol, transcription with all four NTPS was allowed to proceed for 10 min before addition of radiolabel and incubation for 60 min. Reactions labeled aA contained RAP74 and 1 rig/ml a-amanitin. B) Phosphorimager quantitation of the data in "A". 140 Smrew we; 03.; ‘ NS; mom; SN; can; ommrfi o9»; Sm; nappy-w» 's ifir r .. , r- i S 23456789101112 1 13 14 15 16 17 18 19 20 21 22 23 24 Cmrhw we; cg; N54 mom; SN; cam; 0mm; mow; Em; 25 26 27 28 29 30 31 32 33 34 35 36 Figure l 141 DE,DNA,30 74 or mutant _ _ 60 min ACGU" 60 min II I II I m I/ T // I [I ACIGU /U 69/m1n I C I, l 1 O r II I ACU' 59 min // l I // I ll . 1 K II I S G/sarkosyl l8 " F l s: single-round D m: multiple—round -- c: 10min cold-- multiple-round \O 3 +217 transcript (fmol) 0 ON 1409 m 1-217 _m I H” l l l I l I I I l I l\ \o N \o In l\ D v—1 In [\ M [\ .— I: V) M —a r—t I m C I I I I I v— I G '— u—1 w-d H v—I [\ 00 Figure 1 142 (1-205) had a reduced ability to support multiple rounds of transcription after a 10 min incubation with unlabeled NTPS. In the presence of RAP74(1-217) and (1-205), therefore, some transcription factor(s) appeared to become limiting for initiation at later reaction times. In order to characterize new initiation in the extract, the yield of transcripts was determined at different times after addition of NTPS, and transcripts continued to accumulate for more than 90 min (Fig. 2A). The increase in transcription Was due to new initiation rather than slow elongation of chains initiated at earlier times, because when sarkosyl was added to rescue all previously-initiated chains, transcripts nonetheless continued to accumulate for the entire 90 min (open squares). If chains were initiated at early times and slowly elongated, the sarkosyl rescue curve (open squares) would achieve its maximal value at an early time, instead of tracking the curve without sarkosyl (filled squares), as observed. To better understand the activities of RAP74 mutants in multiple round transcription, cycles of transcription were determined as a firnction of time for RAP74 mutants (Figs. 2B and 2C). Mutants for which the graph has a positive slope at the +40 and +80 min time points were judged to be capable of supporting multiple-round transcription. By this criterion, RAP74 was most active followed by RAP74(1-409) and (1-356). RAP74(1-296) had a very weak capacity to support multiple-round transcription, and RAP74(1-217) and (1-205) were inactive. Although defective for multiple-round transcription, RAP74(1-296) and (1-217) were highly active in single- round transcription (Chapter 2). The region of RAP74 between a 409-517 was 143 Figure 2. The central region and the C-terminal domain of RAP74 cooperate to stimulate multiple-round transcription. A) New initiation occurred throughout the 90 min course of the reaction. Assays were done either by the multiple-round protocol (Fig. 5) and stopped at the indicated times (filled squares), or instead of stopping the reactions, sarkosyl was added to block new initiation and transcription continued for an additional 30 min to complete any previously initiated chains (open squares). B and C) Both the central region and the C-terminal domain of RAP74 contributed to multiple-round transcription. Multiple-round transcription was determined using the protocol in Fig. 5, except that reactions were stopped at the indicated times. Single round transcription was estimated using a sarkosyl block procedure with a 79 min elongation. Cycles of transcription were estimated as transcription in the absence (time indicated)/presence of sarkosyl (79 min elongation). 144 ow ease can. 8 ow on I r - 22.2% IT N .; uorrdposucrl 30 soIofig) comes: III: .e 33.8de licl Sm; loll w _§es/ e 8- .i 2. Z O .m cob Tlmx 8558.52. .28.... 8 £29 .3 .eots 8- 25:. s _/ am. n —\ .38.... 2&8 ++ .1- '11 + + + 4.. a; _ u u q o __ .w _u _ N 3 o m _o _n _A ~a —m u _o N_ nu Nu nu No nu ma ~m Figure 2 171 weak affinity for the stalled elongation complex (Price et al., 1989; also see Chapter 3). The addition of ATBE stimulated elongation at both limiting (2 pmol) and saturating (20 pmol) amotmts of TFIIF (lanes 6, 8, 10, and 12). The addition of BSA had no effect (lanes 7, 9, 11, and 13). Because various interactions among these general transcription factors and pol II were previously reported, it was likely that TBP, TFHA, TFIIB, and TFHE stimulated the elongation activity of TFHF by increasing the affinity of TFIIF for the elongation complex. To identify the necessary components for stimulating elongation, various combinations of general transcription factors were tested in the elongation assay (Fig. 2B). 1 pmol of TFIIF did not stimulate elongation above background (compare lanes 3 and 2), while the addition of ATBE slightly enhanced elongation (lanes 19 and 2). However, the addition of ATBE dramatically increased elongation in the presence of 1 pmol TFHF (lanes 4, 3, and 19). The presence of ATBE may recruit TFHF into the elongation complex. Alternatively, ATBE may induce a conformational change in the stalled elongation complex, which favors rapid elongation. Although the presence of all factors gave rise to the strongest activity, none of TBPc, TFIIB, TFHA, or TFHE appeared to be essential for stimulating the elongation activity of TFHF (lanes 4-18). This result suggests that there are redth pathways for recruiting TFHF into the elongation complex albeit with difl‘erent activities. TBPc slightly stimulated elongation in the absence of TFHF but neither TFHA, TFHB, nor TFHE had this capacity (lanes 24- 27). TFHA, TFHB, and TFHE did not affect the ability of TBPc in stimulating elongation (lanes 19-27). 172 Figure 3. TBPc, TFIIA, TFIIB, and TFIIE stimulate the elongation activities of TFIIF and TFIIF mutants. The method was the same as in Fig. 2, except that increasing amounts of TFIIF or TFIIF mutants (l, 5, 20 pmol) were added as indicated. 173 +++ l + +++ l Ammammmm + ATBE F M _ m 500—9 350— 300— . 250_‘ 200— s 150— ‘ 50 Figure 3 174 TBP T T and TFHE stimulate the clan ation activities of TFHF and TFHF mutants Many TFHF mutants were defective in stimulating elongation by pol H (Chapters 2 and 3). Because these factors aided in TFHF recruitment, we wished to test whether TBPc, TFHA, TFHB, and TFHE would rescue these TFHF mutants in elongation or merely function to stabilize these mutants in the complex (Fig. 3). Three TFIIF mutants (1-217, 1176A, M177A) and wild type TFHF (1-517) were tested. 1-217, a TFHF complex containing RAP74(1-217), was fully active in elongation (Chapters 2 and 3; compare lanes 9-11 with 3-5). 1176A and M177A, TFHF complexes each containing a point mutation in RAP74, were highly defective in elongation (Chapter 3; compare lanes 15-17 and 21-23 with 3-5). 1, 5, and 20 pmol of TFHF or TFHF mutant were tested in the presence or the absence of ATBE (TF HA, TBPc, TFHB, and TFHE) in the elongation assay. As shown in Fig. 3, the addition of ATBE increased the elongation activities of TFHF and all three TFHF mutants. Although 1176A and M177A were stimulated by the addition of ATBE, the activities of both mutants remained very low (lanes 15-26). Therefore, the intrinsic defects of 1176A and M17 7A in elongation could not be fully rescued by the general factors. It was previously demonstrated that the C-terminal region of RAP74 (aa 358-517) interacted with TFHB in vitro (Fang and Burton, 1996), however, this interaction was dispensable for ATBE to stimulate the elongation activity of TFIIF because both 1-517 and 1-217 was stimulated by ATBE to a similar extent (compare lanes 12-14 with lanes 6-8). This was consistent with the result that TFIIB was not essential for stimulating the 175 elongation activity of TFIIF (lane 7, Fig. 2B). Other contacts among TBPc, TFHA, TFHB, TFHE, and TFHF presumably helped to recruit TF HF into the elongation complex. 176 "ll-1 Discussion RAP74 in initiation Both RAP30 and RAP74 were required for transcription from a linear template in the HeLa extract (Lei et al., 1998) and in a defined system consisting of TBP, TFHB, TFHE, TFIH-I, and pol H (Tan et al., 1994). However, RAP30 alone was sufficient to support transcription flour a supercoiled template when supplemented with pol H, TBP, and TFHB (Tyree et al., 1993). RAP74 was not essential but stimulated the level of transcription. In order to understand the different requirements for RAP74, a defined system was established with recombinant human TBPc, TFHB, TFHA, TFIIE, TFHF, pmified calf thymus pol H, and a negatively supercoiled template pML(C2AT)A71 (Fig. 1). Clearly, pol H, TBPc, TFHB, TFHE, and TFHF were sufficient for initiation in this system. Consistent with previous results (Tyree et al., 1993), the addition of RAP30 instead of TFHF indeed supported a minimal level of transcription (lanes 14 and 15). Although RAP74 alone was inactive (lanes 16 and 17), the presence of RAP74 strongly stimulated the level of transcription (compare lanes 11 and 14). Because RAP74 was required. for transcription from a linear template but not from a supercoiled template, the negative supercoils in the DNA template apparently relieved the requirement of RAP7 4 in initiation. This result is not lmexpected because RAP74 helps to induce DNA wrapping around pol H and isomerization of the preinitiation complex. Similar to DNA wrapping, negative supercoiling may cause a torsional strain on the template to facilitate formation of the open complex, thus bypassing the requirement for RAP74 in template isomerization. The addition of RAP74 may induce DNA wrapping on the supercoiled template and stimulate transcription by further enhancing isomerization. RAP74 is 177 necessary for initiation fiom a linear template (no superhelical tension) because under this condition the DNA wrapping becomes essential for isomerization. A similar observation was made for TFHE. TF HE is necessary for transcription from a linear template but partially dispensable for transcription flour a supercoiled template (lane 9). The requirement of TFHE in the defined system depends on the stability of the DNA template (Holstege et al., 1995). Under conditions such as low salt in which DNA strand separation is favored, TFHE weakly stimulates transcription. Under conditions in which DNA strand separation is more difficult, TFHE strongly stimulates transcription. These observations led to the conclusion that TFIIE plays a direct role in open complex formation. It remains important to analyze RAP74 in the defined system under different conditions to test whether the requirement for RAP74 is directly related to the stability of the double helix. Human TBPc stimulates elongation by ml H TBP is a universal transcription factor required for accurate initiation by all three eukaryotic RNA polymerases (Hernandez, 1993). The primary function of TBP in pol H transcription is to bind the TATA element and nucleate the assembly of the PIC. TBPc, the C-terminal core of TBP, is sufficient for PIC assembly and initiation (Orphanides et al., 1996). It is generally believed that TBP remains bound to the TATA element when pol H exits fiorn the promoter and begins elongation, although the evidence in support of this view is insufficient. Fig. 2 demonstrated, for the first time, that human TBPc did stimulate elongation when added to the stalled elongation complex which was washed fiee of accessory factors. Although the stimulation by TBPc is weak, it is highly reproducible. Neither BSA, TFHA, TFHB, nor TFIIE stimulates elongation by pol H. 178 Because the elongation complex is stalled near the start site, it is possible that TBPc binds the TATA element and induces a conformational change in the stalled elongation complex that favors rapid elongation. It remains important to test whether TBPc or full length TBP can stimulate elongation independent of a TATA element. Several approaches are proposed: 1) stall the elongation complex farther downstream fiom the start site and test whether TBPc or TBP can stimulate elongation; 2) stall the elongation complex, remove the upstream DNA sequence including the TATA element by restriction digestion and then test whether TBPc or TBP can stimulate elongation; 3) test whether TBP mutants that are defective for DNA binding can stimulate elongation. Genegl transcription factors stimulate the elongation am of TFHF TBPc, TFHB, TFHA, and TF HE stimulate the elongation activity of TFHF (Fig. 2). None of these factors (TBPc, TFHB, TFHA, and TFHE; “ATBE”) is essential for the stimulation but the addition of all four factors gives rise to the strongest stimulation observed. TFHF has been shown to have a weak affinity for the stalled elongation complex. Therefore, TBPc, TFHB, TFHA, and TFIIE may recruit TFIIF into the elongation complex through protein-protein contacts and possibly protein-DNA contacts. These general factors may cooperate with TFHF to induce DNA wrapping in the elongation complex as they do in the preinitiation complex (Robert et al., 1998). DNA wrapping is proposed to maintain the elongation complex in an isomerized state that is essential for phosphodiester bond formation. . TBPc, TFHB, TFHA, and TFIIE also stimulate the elongation activities of several TFHF mutants tested (Fig. 3). The C-terminal domain and the central region of RAP74 are dispensable for stimulation by ATBE, because TFIIF complexes containing either 179 wild type RAP74 or RAP74(1-217) are stimulated to the same extent. Il76A and M177A are highly defective in elongation but both can be stimulated by ATBE. However, the activities of Il76A and M177A in the presence of ATBE remain low. This demonstrates that the intrinsic defects of 117 6A and M177A in elongation can not be rescued by ATBE, suggesting that recruitment of TFIIF mutants into elongation complex is not suficient to stimulate rapid elongation. This report demonstrates for the first time the novel activities of TBP, TFIIB, TFIIA, and TFIIE in transcript elongation. These general factors may recruit TFHF and possibly help wrap the DNA template in the elongation complex. It remains important to test whether TBP, TFHB, TFIIA, TFHE stay in the elongation complex or they simply recruit TFIIF into the elongation complex and subsequently release. These studies strongly suggest that factors that have previously been considered to be only involved in initiation are very likely to have roles in elongation as well. It will be important to determine whether TBP, TFIIB, TFIIA, and TFIIE associate with elongation complexes in vivo. 180 Acknowledgments I thank Drs. Delin Ren, Ann Finkelstein, Sean Juo, Jim Geiger, Steve Triezenberg, Fan Shen for proteins and Danny Reinberg for TFIIE clones. This work was supported by a grant fi'om the American Cancer Society, by Michigan State University, and by the Michigan State University Agricultural Experiment Station. 181 FUTURE RESEARCH The functional domains of RAP74 have been mapped in this study. The N- terminal, domain of RAP74 is important for both initiation and elongation stimulation. Several amino acid residues in a highly conserved region of the N-terminal domain have been formd to be most critical for both initiation and elongation. These results suggest that RAP74 plays an identical role in both initiation and elongation. RAP74 is proposed to induce DNA wrapping in both the preinitiation complex and the elongation complex to facilitate isomerization. The C-terminal domain and the central region of RAP74 do not contribute strongly to single-round initiation or elongation stimulation but do stimulate multiple-round transcription. Additionally, a novel ftmction for TBP, TFIIB, TFHA, and TFIIE in elongation is demonstrated. These factors, previously considered to be involved only in initiation, stimulate the elongation activity of TFHF, apparently by recruiting TFHF into the elongation complex. TBPc alone slightly stimulates elongation by pol H. Several projects can be developed fiom these results to further improve our understanding of the mechanisms of RAP74 in transcriptional initiation, elongation, and recycling. RAP74 is proposed to facilitate formation of the open complex by inducing DNA wrapping in initiation. A series of site-directed mutants of RAP74 has been constructed and many have reduced activities in initiation. ‘ However, even the most defective mutants of RAP74 are capable of binding RAP30 and delievering pol II to the preinitiation complex. RAP74 mutants that are defective in initiation need to be tested in the photocrosslinking assay to determine whether these mutants are defective in DNA 182 wrapping, and these tests are in progress in collaboration with Dr. Benoit Coulombe and his laboratory. How TFHF stimulates the elongation rate of pol H is not yet known. Detailed studies of TFHF in the elongation complex may face significant technical difficulties. By analogy, RAP74 is pr0posed to induce DNA wrapping in the elongation complex as RAP74 does in the preinitiation complex. The elongation complex at any template position may have multiple configurations. DNA wrapping is expected to maintain pol H in the isomerized state that is active in phosphodiester bond formation. RAP74 mutants that are defective in elongation stimulation are expected to be defective in maintaining the elongation complex in the isomerized state. Plasmids pML20-42 and pML20—49 containing the adenovirus major late promoter with modified sequences were kindly provided by Dr. Donal Luse. Immobilized templates containing the modified sequences were prepared and tested to be active in transcription assays (data not shown). These immobilized templates will be used to generate homogenous populations of stalled elongation complexes. Several approaches are designed to analyze the efi‘ect of RAP74 on the elongation complex. First, RAP74 can be tested for the ability to affect the DNA boundary of the elongation complex. DNA wrapping is expected to increase the extent of DNA-protein contact within the elongation complex particularly in the downstream direction. Preliminary results demonstrated that TFIIF did not affect the DNA boundary as mapped by exo III footprinting (personal communications with Dr. Donal Luse). However, TFIIF has a weak affinity for the stalled complex and may not bind tightly enough to induce changes in the footprint. Because TBPc, TFIIB, TFIIA, and TFIIE appear to recruit TFHF into the elongation complex, the DNA boundary of the elongation 183 complex will be mapped in the presence or the absence of TFIIF, TBPc, TFIIB, TFHA, and TFIIE. Second, RAP74 can be tested for the ability to afi‘ect the transcription bubble in the elongation complex by KMnO4 footprinting. DNA wrapping is expected to stabilize the open complex and therefore enhance the intensity of the KMnO4 footprint. Furthermore, RAP74 may affect the Km of the NTP substrates in elongation. This can be tested by analyzing the elongation rate in the presence or the absence of RAP74 and under various NTP concentrations. RAP74 mutants that are defective in elongation stimulation will be used as controls. General factors TBPc, TFHB, TFIIA, and TFHE apparently stimulate the elongation activity of TFIIF by recruiting TFIIF into the elongation complex. This may be directly demonstrated by analyzing the elongation complex in an RNA gel mobility shift assay. Synchronously stalled elongation complexes will be made using the immobilized templates containing the modified DNA sequence, and the accessory factors will be removed by washing with 1% sarkosyl and 0.5 M KCl. TFHF or TFIIF mutants will be added in the presence or the absence of general factors. After a brief incubation, the flanking DNA sequence will be removed by restriction enzyme digestion and the elongation complexes will be analyzed on a native polyacrylamide gel. The radioactive labeled RNA transcript will serve to identify the complex. This approach may also demonstrate whether general factors remain in the elongation complex because different shifts will be observed if general factors are present. . TFIIF, TFIIB, and CTD phosphatase are proposed to be components of a transcriptional recycling apparatus that regulates the conversion of pol 110 to pol IIA. TFIIF, TFIIB, and FCP-l, an essential component of CTD phosphatase, are all present in 184 the pol 11 holoenzyme prepared fiom HeLa extracts using GST-SII as an affinity ligand. It will be interesting to test whether this holoenzyme is capable of transcriptional recycling. If it does recycle, the pol II holoenzyme can be used to identify additional factors important for transcriptional recycling by the method of fi‘actionation and reconstitution. It will also be easier to track the phosphorylation status of the CTD in the pol II holoenzyme than HeLa extracts because of higher background in the extracts. The phosphorylation status of the CTD will be analyzed in the presence of RAP74 or RAP74 mutants to determine whether the activities of RAP74 mutants in supporting multiple- round transcription coorelate directly with the extent of CTD dephosphorylation. A GST-SII clone has been kindly provided by Francois Robert with the permission of Dr. Jack Greenblatt. The three-dimensional structures of TBP, TBP-TATA complex, TFIIB-TBP- DNA complex, and TFIIA-TBP-DNA complex have provided many insights into the fimctions and mechanisms of these transcription factors. The three-dimensional structure of the DNA binding domain of RAP30 was recently solved by NMR. However, no three- dimensional struCture of RAP74 has been reported. We are currently in collaboration with Dr. Jim Geiger and his colleagues to determine the structmes of RAP74 and TFIIF using X-ray crystallography techniques. During the course of this work, several in vitro assays have been established for studying the functions of RAP74. These include the gel mobility shift assay, various initiation assays (pulse-spin, pulse-chase, and pulse—sarkosyl chase), a sensitive elongation stimulation assay, and a recycling assay. These assays will be useful in analyzing new RAP74 mutants and other transcription factors such as RAP30. 185 REFERENCES Adamczewski, J. P., Rossignol, M., Tassan, J. P., Nigg, E. A., Moncollin, V., and Egly, J. M. (1996). MAT] , cdk7 and cyclin H form a kinase complex which is UV light-sensitive upon association with TFIIH. EMBO J 15, 1877-84. Agarwal, K., Back, K. H., Jeon, C. J., Miyamoto, K., Ueno, A., and Yoon, H. S. (1991). Stimulation of transcript elongation requires both the zinc finger and RNA polymerase II binding domains of human TFIIS. Biochemistry 30, 7842-7851. Agostini, I., Navarro, J. M., Rey, F., Bouhamdan, M., Spire, B., Vigne, R., and Sire, J. (1996). The human immunodeficiency virus type 1 Vpr transactivator: cooperation with promoter-bound activator domains and binding to TFIIB. J Mol Biol 261, 599-606. Akoulitchev, S., Makela, T. P., Weinberg, R. A., and Reinberg, D. (1995). Requirement for TFIIH kinase activity in transcription by RNA polymerase II. Nature 377, 557-60. Alland, L., Muhle, R., Hou, H., Jr., Potes, J ., Chin, L., Schrerher-Agus, N., and DePinho, R. A. (1997). Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression. Natm'e 387, 49-55. Apone, L. M., Virbasius, c. M., Reese, J. C., and Green, M. R. (1996). Yeast TAF(II)90 is required for cell-cycle progression through GZ/M but not for general transcription activation. Genes Dev 10, 2368-80. Arany, 2., Sellers, W. R., Livingston, D. M., and Eckner, R. (1994). BIA-associated p300 and CREE-associated CBP belong to a conserved family of coactivators. Cell 77, 799-800. ’ Archambault, J., Lacroute, F., Ruet, A., and Friesen, J. D. (1992). Genetic interaction between transcription elongation factor TFHS and RNA polymerase 11. Mol Cell Biol 12, 4142-41 52. Arias, J. A., and Dynan, W. S. (1989). Promoter-dependent transcription by RNA polymerase 11 using immobilized enzyme complexes. J Biol Chem 264, 3223-3229. Arnosti, D. N., Merino, A., Reinberg, D., and Schaffiaer, W. (1993). Oct-2 facilitates functional preinitiation complex assembly and is continuously required at the promoter for multiple rounds of transcription. EMBO J 12, 157-66. Aso, T., Conaway, J. W., and Conaway, R. C. (1994). Role of core promoter structure in assembly of the RNA polymerase II preinitiation complex. A common pathway for formation of preinitiation intermediates at many TATA and TATA-less promoters. J Biol Chem 269, 26575-26583. 186 Aso, T., Haque, D., Barstead, R. J., Conaway, R. C., and Conaway, J. W. (1996). The inducible elongin A elongation activation domain: structure, fimction and interaction with the elongin BC complex. EMBO J 15, 5557-66. Aso, T., Lane, W. S., Conaway, J. W., and Conaway, R. C. (1995). Elongin ($111): a multisubunit regulator of elongation by RNA polymerase II. Science 269, 1439-1443. Aso, T., Vasavada, H. A., Kawaguchi, T., Germino, F. J., Ganguly, s., Kitajima, s., Weissman, S. M., and Yasukochi, Y. (1992). Characterization of cDNA for the large subunit of the transcription initiation factor TFHF. Nature 355, 461-4. Asttuias, F. J., Meredith, G. D., Poglitsch, C. L., and Kornberg, R. D. (1997). Two conformations of RNA polymerase 11 revealed by electron crystallography. J Mol Biol 272, 536-540. Auble, D. T., and Hahn, S. (1993). An ATP-dependent inhibitor of TBP binding to DNA. Genes Dev 7, 844-56. Auble, D. T., Hansen, K. E., Mueller, C. G., Lane, W. S., Thomer, J ., and Hahn, S. (1994). Motl, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism. Genes Dev 8, 1920-34. Auble, D. T., Wang, D., Post, K. W., and Hahn, S. (1997). Molecular analysis of the SNF2/ SW12 protein family member MOT] , an ATP- driven enzyme that dissociates TATA-binding protein fiom DNA. Mol Cell Biol 1 7, 4842-4851. Awrey, D. E., Shimasaki, N., Koth, C., Weilbaecher, R., Olmsted, V., Kazanis, S., Shari, X., Arellano, J ., Arrowsmith, C. H., Kane, C. M., and Edwards, A. M. (1998). Yeast transcript elongation factor (TFHS), structure and flmction. 11: RNA polymerase binding, transcript cleavage, and read-through. J Biol Chem 273, 22595-22605. Awrey, D. E., Weilbaecher, R. G., Hemming, S. A., Orlicky, S. M., Kane, C. M., and Edwards, A. M. (1997). Transcription elongation through DNA arrest sites. A multistep involving both RNA polymerase II subunit RPB9 and TFIIS. J Biol Chem 272, 14747-14754. Bagby, S., Kim, S., Maldonado, E., Tong, K. 1., Reinberg, D., and Ikura, M. (1995). Solution structure of the C-terminal core domain of human TFIIB: similarity to cyclin A and interaction with TATA-binding protein. Cell 82, 857-67 . Barberis, A., Muller, C. W., Harrison, S. C., and Ptashne, M. (11993). Delineation of two ftmctional regions of transcription factor TFIIB. Proc Natl Acad Sci U S A 90, 5628-32. 187 Barberis, A., Pearlberg, J., Simkovich, N., Farrell, S., Reinagel, P., Bamdad, C., Sigal, G., and Ptashne, M. (1995). Contact with a component of the polymerase 11 holoenzyme sufices for gene activation. Cell 81, 359-68. Barlev, N. A., Candau, R., Wang, L., Darpino, P., Silverman, N., and Berger, S. L. (1995). Characterization of physical interactions of the putative transcriptional adaptor, ADA2, with acidic activation domains and TATA- binding protein. J Biol Chem 270, 19337-19344. Bartholomew, B., Dahmus, M. E., and Meares, C. F. (1986). RNA contacts subunits Ho and He in HeLa RNA polymerase II transcription complexes. J Biol Chem 261 , 14226- 1423 l . Bengal, E., Flores, 0., Krauskopf, A., Reinberg, D., and Aloni, Y. (1991). Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase H. Mol Cell Biol 11, 1195-206. Bentley, D. L. (1995). Regulation of transcriptional elongation by RNA polymerase II. Curr Opin Genet Dev 5, 210-216. Berger, S. L., Cress, W. D., Cress, A., Triezenberg, S. J ., and Guarente, L. (1990). Selective inhibition of activated but not basal transcription by the acidic activation domain of VP16: evidence for transcriptional adaptors. Cell 61, 1199-1208. Berger, S. L., Pina, B., Silverman, N., Marcus, G. A., Agapite, J ., Regier, J. L., Triezenberg, S. J ., and Guarente, L. (1992). Genetic isolation of ADA2: a potential transcriptional adaptor required for firnction of certain acidic activation domains. Cell 70, 25 1-265. Berroteran, R. W., Ware, D. E., and Hampsey, M. (1994). The sua8 suppressors of Saccharomyces cerevisiae encode replacements of conserved residues within the largest subunit of RNA polymerase II and affect transcription start site selection similarly to sua7 (TFIIB) mutations. Mol Cell Biol 14, 226-37. Blanco, J. C., Wang, 1. M., Tsai, S. Y., Tsai, M. J ., BW, O. M., Jurutka, P. W., Haussler, M. R., and Ozato, K. (1995). Transcription factor TFIIB and the vitamin D receptor cooperatively activate ligand-dependent transcription. Proc Natl Acad Sci U S A 92, 1535-9. . Blau, J., Xiao, H., McCracken, S., O'Hare, P., Greenblatt, J., and Bentley, D. (1996). Three functional classes of transcriptional activation domain. Mol Cell Biol 16, 2044- 2055. BorukhOv, S., Polyakov, A., Nikiforov, V., and Goldfarb, A. (1992). GreA protein: a transcription elongation factor from Escherichia coli. Proc Natl Acad Sci U S A 89, 8899- 902. 188 Borukhov, S., Sagitov, V., and Goldfarb, A. (1993). Transcript cleavage factors fi'om E. coli. Cell 72, 459-66. Bradsher, J. N., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1993). RNA polymerase II transcription factor SHI. 1. Identification, purification, and properties. J Biol Chem 268, 25587-25593. Bradsher, J. N., Tan, 8., McLaury, H. J ., Conaway, J. W., and Conaway, R. C. (1993). RNA polymerase II transcription factor $111. 11. Functional properties and role in RNA chain elongation. J Biol Chem 268, 25594—25603. Brown, S. A., Imbalzano, A. N., and Kingston, R. E. (1996). Activator-dependent regulation of transcriptional pausing on nucleosomal templates. Genes Dev 10, 1479-90. Brownell, J . E., Zhou, J ., Ranalli, T., Kobayashi, K, Edmondson, D. G., Roth, S. Y., and Allis, C. D. (1996). Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell 84, 843-851. Buermeyer, A. B., Strasheim, L. A., McMahon, S. L., and Farnham, P. J. (1995). Identification of cis-acting elements that can obviate a requirement for the C-terminal domain of RNA polymerase II. J Biol Chem 2 70, 67 98-807. Bunick, D., Zandomeni, R., Ackerman, S., and Weinmann, R. (1982). Mechanism of RNA polymerase II—specific initiation of transcription in vitro: ATP requirement and lmcapped runoff transcripts. Cell 29, 877-886. Buratowski, S., Hahn, S., Guarente, L., and Sharp, P. A. (1989). Five intermediate complexes in transcription initiation by RNA polymerase 11. Cell 56, 549-61. Btu'atowski, S., and Zhou, H. (1993). Functional domains of transcription factor TFIIB. Proc Natl Acad Sci U S A 90, 5633-7. Burke, T. W., and Kadonaga, J. T. (1997). The downstream core promoter element, DPE, is conserved fi'om Drosophila to humans and is recognized by TAF 1160 of Drosophila. Genes Dev 11, 3020-3031. Burke, T. W., and Kadonaga, J. T. (1996). Drosophila TFIID binds to a conserved downstream basal promoter element that is present in many TATA-box-deficient promoters. Genes Dev 10, 711-24. Burley, S. K., and Roeder, R. G. (1996). Biochemistry and structural biology of transcription factor IID (TFIID). Annu Rev Biochem 65, 769-99. Burns, L. G., and Peterson, C. L. (1997). Protein complexes for remodeling chromatin. Biochim Biophys Acta 1350, 159-168. 189 Bluton, Z. P., Killeen, M., Sopta, M., Ortolan, L. G., and Greenblatt, J. (1988). RAP30/74: a general initiation factor that binds to RNA polymerase 11. Mol Cell Biol 8, 1602-13. Btuton, Z. P., Ortolan, L. G., and Greenblatt, J. (1986). Proteins that bind to RNA polymerase II are required for accurate initiation of transcription at the adenovirus 2 major late promoter. EMBO J 5, 2923-30. Cairns, B. R, Henry, N. L., and Kornberg, R. D. (1996). TFG/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukemogenic proteins ENL and AF-9. Mol Cell Biol 16, 3308-16. Cairns, B. R., Kim, Y. J., Sayre, M. H., Laurent, B. C., and Kornberg, R. D. (1994). A multisubunit complex containing the SWIl/ADR6, SWIZ/SNF2, SW13, SNF5, and SNF6 gene products isolated from yeast. Proc Natl Acad Sci U S A 91, 1950-4. Cairns, B. R., Lorch, Y., Li, Y., Zhang, M., Lacomis, L., Erdjument Bromage, H., Tempst, P., Du, J., Lament, B., and Kornberg, R. D. (1996). RSC, an essential, abundant chromatin-remodeling complex. Cell 87, 1249-60. Candau, R., and Berger, S. L. (1996). Structural and functional analysis of yeast putative adaptors. Evidence for an adaptor complex in vivo. J Biol Chem 271, 5237-5245. Candau, R., Moore, P. A., Wang, L., Barlev, N., Ying, C. Y., Rosen, C. A., and Berger, S. L. (1996). Identification of hmnan proteins flmctionally conserved with the yeast putative adaptors ADA2 and GCNS. Mol Cell Biol 16, 593-602. Candau, R., Zhou, J. X., Allis, C. D., and Berger, S. L. (1997). Histone acetyltransferase activity and interaction with ADA2 are critical for GCNS function in vivo. Embo J 16, 555-565. Carmen, A. A., Rundlett, S. E., and Grunstein, M. (1996). HDAl and HDA3 are components of a yeast histone deacetylase (HDA) complex. J Biol Chem 271, 15837- 15844. Carrozza, M. J ., and DeLuca, N. A. (1996). Interaction of the viral activator protein ICP4 with TFIID through TAF250. Mol Cell Biol 16, 3085-93. ‘ Chambers, R. S., and Dahmus, M. E. (1994). Purification and characterization of a phosphatase fi'om HeLa cells which dephosphorylates the C-terminal domain of RNA polymerase II. J Biol Chem 269, 26243-26248. Chambers, R. S., Wang, B. Q., Burton, Z. P., and Dahmus, M. E. (1995). The activity of COOH-terminal domain phosphatase is regulated by a docking site on RNA polymerase II and by the general transcription factors [IF and IIB. J Biol Chem 270, 14962-9. 190 Chang, J ., Kim, D. H., Lee, S. W., Choi, K. Y., and Sung, Y. C. (1995). Transactivation ability of p53 transcriptional activation domain is directly related to the binding affinity to TATA-binding protein. J Biol Chem 270, 25014-9. Chao, D. M., Gadbois, E. L., Murray, P. J., Anderson, S. F., Sonu, M. S., Parvin, J. D., and Young, R. A. (1996). A mammalian SRB protein associated with an RNA polymerase H holoenzyme. Nature 380, 82-5. Chatterjee, S., and Struhl, K. (1995). Connecting a promoter-bound protein to TBP bypasses the need for a transcriptional activation domain. Nature 374, 820-822. Chen, H., Lin, R. J ., Schiltz, R. L., Chakravarti, D., Nash, A., Nagy, L., Privalsky, M. L., Nakatani, Y., and Evans, R M. (1997). Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90, 569-580. Chen, J. L., Attardi, L. D., Verrijzer, C. P., Yokomori, K., and Tjian, R. (1994). Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators. Cell 79, 93-105. Chi, T., and Carey, M. (1996). Assembly of the isomerized TFIIA—TFIID--TATA ternary complex is necessary and sufficient for gene activation. Genes Dev 10, 2540-50. Chi, T., and Carey, M. (1993). The ZEBRA activation domain: modular organization and mechanism of action. Mol Cell Biol 13, 7045-55. Chiang, c. M., and Roeder, R. o. (1995). Cloning ofan intrinsic human TFHD subunit that interacts with multiple transcriptional activators. Science 267, 531-6. Cho, H., Orphanides, G., Sun, X., Yang, X. J .,‘Ogryzko, V., Lees, E., Nakatani, Y., and Reinberg, D. (1998). A human RNA polymerase II complex containing factors that modify chromatin structure. Mol Cell Biol 18, 5355-5363. Chodosh, L. A., Fire, A., Samuels, M., and Sharp, P. A. (1989). 5,6-Dichloro-1-beta-D- ribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase II in vitro. J Biol Chem 264, 2250-7. Choy, B., and Green, M. R. (1993). Eukaryotic activators flmction during multiple steps of preinitiation complex assembly. Nature 366, 531-6. Cisek, L. J., and Corden, J. L. (1989). Phosphorylation of RNA polymerase by the murine homologue of the cell- cycle control protein cdc2. Nature 339, 679-684. Cisrnowski, M. J ., Lafi‘, G. M., Solomon, M. J ., and Reed, S. I. (1995). KIN28 encodes a C-terminal domain kinase that controls mRNA transcription in Saccharomyces cerevisiae 191 but lacks cyclin-dependent kinase-activating kinase (CAK) activity. Mol Cell Biol 15, 2983-92. Clark, A. R., and Docherty, K. (1993). Negative regulation of transcription in eukaryotes. Biochem J 296, 521-541. Cochet-Meilhac, M., Nuret, P., Com'valin, J. C., and Chambon, P. (1974). Animal DNA- dependent RNA polymerases. 12. Determination of the cellular number of RNA polymerase B molecules. Biochim Biophys Acta 353, 185-192. Collart, M. A. (1996). The NOT, SPT3, and MOT 1 genes functionally interact to regulate transcription at core promoters. Mol Cell Biol 16, 6668-76. Collart, M. A., and Struhl, K. (1994). NOT1(CDC39), NOT2(CDC36), NOT3, and NOT4 encode a global-negative regulator of transcription that differentially affects TATA-element utilization. Genes Dev 8, 525-37. Comai, L., Tanese, N., and Tjian, R. (1992). The TATA-binding protein and associated factors are integral components of the RNA polymerase I transcription factor, SL1. Cell 68, 965-76. Conaway, J. W., Kamura, T., and Conaway, R. C. (1998). The Elongin BC complex and the von Hippel-Lindau tumor suppressor protein. Biochim Biophys Acta 1 3 7 7, M49- M54. Conaway, R. C., and Conaway, J. W. (1993). General initiation factors for RNA polymerase II. Annu Rev Biochem 62, 161-90. Conaway, R. C., and Conaway, J. W. (1997). General transcription factors for RNA polymerase II. Prog Nucleic Acid Res Mol Biol 56, 327-346. Conaway, R. C., and Conaway, J. W. (1989). An RNA polymerase II transcription factor has an associated DNA-dependent ATPase (dATPase) activity strongly stimulated by the TATA region of promoters. Proc Natl Acad Sci U S A 86, 7356-60. Conaway, R. C., Garrett, K P., Hanley, J. P., and Conaway, J. W. (1991). Mechanism of promoter selection by RNA polymerase II: mammalian transcription factors alpha and beta gamma promote entry of polymerase into the preinitiation complex. Proc Natl Acad Sci U S A 88, 6205-9. Cormack, B. P., and Struhl, K. (1992). The TATA-binding protein is required for transcription by all three nuclear RNA polymerases in yeast cells. Cell 69, 685-96. Cote, J ., Quinn, J., Workman, J. L., and Peterson, C. L. (1994). Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science 265 , 53-60. ' 192 Coulombe, B., Li, J ., and Greenblatt, J. (1994). Topological localization of the human transcription factors IIA, IIB, TATA box-binding protein, and RNA polymerase H- associated protein 30 on a class 11 promoter. J Biol Chem 269, 19962-7. Craig, M. L., Sub, W. C., and Record, M. T., Jr. (1995). H0. and DNase Iprobing of E sigma 70 RNA polymerase—lambda PR promoter open complexes: Mg2+ binding and its structural consequences at the transcription start site. Biochemistry 34, 15624-15632. Dahmus, M. E. (1995). Phosphorylation of the C-terminal domain of RNA polymerase II. Biochim Biophys Acta 1261, 171-182. Dahmus, M. E. (1996). Reversible phosphorylation of the C-terminal domain of RNA polymerase II. J Biol Chem 271, 19009-19012. Dahmus, M. E. (1994). The role of multisite phosphorylation in the regulation of RNA polymerase II activity. Prog Nucleic Acid Res Mol Biol 48, 143-179. Damania, B., Lieberman, P., and Alwine, J. C. (1998). Simian virus 40 large T antigen stabilizes the TATA-binding protein- TFIIA complex on the TATA element. Mol Cell Biol 18, 3926-3935. Darst, S. A., Edwards, A. M., Kubalek, E. W., and Kornberg, R. D. (1991). Three- dimensional structm'e of yeast RNA polymerase II at 16 A resolution. Cell 66, 121-8. Darst, S. A., Kubalek, E. W., and Kornberg, R. D. (1989). Three-dimensional structure of Escherichia coli RNA polymerase holoenzyme determined by electron crystallography. Nature 340, 730-2. Defossez, P. A., Baert, J. L., Monnot, M., and de Launoit, Y. (1997). The ETS family member ERM contains an alpha-helical acidic activation domain that contacts TAF 1160. Nucleic Acids Res 25, 4455-4463. DeJong, J ., Bernstein, R., and Roeder, R. G. (1995). Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription. Proc Natl Acad Sci U S A 92, 3313-7. DeJong, J ., and Roeder, R. G. (1993). A single cDNA, hTFIIA/alpha, encodes both the p35 and p19 subunits of human TFIIA. Genes Dev 7, 2220-34. Dikstein, R., Ruppert, S., and Tjian, R. (1996). TAFII250 is a bipartite protein kinase that phosphorylates the base transcription factor RAP74. Cell 84, 781-90. Drapkin, R., Le Roy, G., Cho, H., Akoulitchev, S., and Reinberg, D. (1996). Human cyclin-dependent kinase-activating kinase exists in three distinct complexes. Proc Natl Acad Sci U S A 93, 6488-93. 193 Drapkin, R., Reardon, J. T., Ansari, A., Huang, J. C., Zawel, L., Ahrr, K., Sancar, A., and Reinberg, D. (1994a). Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase 11. Nature 368, 769-72. Drapkin, R., and Reinberg, D. (1994b). The multifrmctional TFIIH complex and transcriptional control. Trends Biochem Sci 19, 504-8. Drapkin, R., Sancar, A., and Reinberg, D. (19940). Where transcription meets repair. Cell 7 7, 9-12. Duan, D. R., Pause, A., Burgess, W. H., Aso, T., Chen, D. Y., Garrett, K. P., Conaway, R. C., Conaway, J. W., Linehan, W. M., and Klausner, R. D. (1995). Inhibition of transcription elongation by the VHL tumor suppressor protein. Science 269, 1402-1406. Dvir, A., Conaway, R. C., and Conaway, J. W. (1997). A role for TFIIH in controlling the activity of early RNA polymerase II elongation complexes. Proc Natl Acad Sci U S A 94, 9006-9010. Dvir, A., Garrett, K. P., Chalut, C., Egly, J. M., Conaway, J. W., and Conaway, R. C. (1996). A role for ATP and TFIIH in activation of the RNA polymerase II preinitiation complex prior to transcription initiation. J Biol Chem 271, 7245-7248. Dynlacht, B. D., Hoey, T., and Tjian, R. (1991). Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation. Cell 66, 563-7 6. Edwards, A. M., Kane, C. M., Young, R. A., and Kornberg, R. D. (1991). Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro. J Biol Chem 266, 71-5. Eick, D., Wedel, A., and Hermann, H. (1994). From initiation to elongation: comparison of transcription by prokaryotic and eukaryotic RNA polymerases. Trends Genet 10, 292- 296. Eisenmann, D. M., Dollard, C., and Winston, F. (1989). SPT15, the gene encoding the yeast TATA binding factor TFIID, is required for normal transcription initiation in vivo. Cell 58, 1 183-91. Emili, A., Greenblatt, J ., and Ingles, C. J. (1994). Species-specific interaction of the glutamine-rich activation domains of Spl with the TATA box-binding protein. Mol Cell Biol 14, 1582-93. Fang, S..M., and Bmton, Z. F. (1996). RNA polymerase H-associated protein (RAP) 74 binds transcription factor (TF) IIB and blocks TFIIB-RAP30 binding. J Biol Chem 271, 1 1703-9. 194 Farrell, S., Simkovich, N., Wu, Y., Barberis, A., and Ptashne, M. (1996). Gene activation by recruitment of the RNA polymerase H holoenzyme. Genes Dev 10, 2359-67. Feaver, W. J ., Gileadi, 0., Li, Y., and Kornberg, R. D. (1991). CTD kinase associated with yeast RNA polymerase H initiation factor b. Cell 67, 1223-30. Feaver, w. J., Henry, N. L., Bushnell, D. A., Sayre, M. H., Brickner, J. H., Gileadi, o., and Kornberg, R. D. (1994). Yeast TFHE. Cloning, expression, and homology to vertebrate proteins. J Biol Chem 269, 27549-53. Feaver, W. J ., Svejstrup, J. Q., Bardwell, L., Bardwell, A. J ., Buratowski, S., Gulyas, K. D., Donahue, T. F., Friedberg, E. C., and Kornberg, R. D. (1993). Dual roles of a multiprotein complex fi'om S. cerevisiae in transcription and DNA repair. Cell 75, 1379- 87. Feaver, W. J., Svejstrup, J. Q., Henry, N. L., and Kornberg, R. D. (1994). Relationship of CDK-activating kinase and RNA polymerase H CTD kinase TFHH/TFIHC Cell 79, 1103- 9. Finkelstein, A., Kostrub, C. R, Li, J., Chavez, D. P., Wang, B. Q., Fang, S. M., Greenblatt, J., and Burton, Z. F. (1992). A cDNA encoding RAP74, a general initiation factor for transcription by RNA polymerase H. Nature 355, 464-7. Flores, 0., Ha, 1., and Reinberg, D. (1990). Factors involved in specific transcription by mammalian RNA polymerase H. Purification and submit composition of transcription factor HF. J Biol Chem 265, 5629-34. Flores, 0., Lu, H., Killeen, M., Greenblatt, J., Burton, Z. P., and Reinberg, D. (1991). The small subunit of transcription factor HF recruits RNA polymerase H into the preinitiation complex. Proc Natl Acad Sci U S A 88, 9999-10003. Flores, 0., Lu, H., and Reinberg, D. (1992). Factors involved in specific transcription by mammalian RNA polymerase H. Identification and characterization of factor IIH. J Biol Chem 267, 2786-93. Flores, 0., Maldonado, E., Burton, 2., Greenblatt, J., and Reinberg, D. (1988). Factors involved in specific transcription by mammalian RNA polymerase H. RNA polymerase H-associating protein 30 is an essential component of transcription factor IIF. J Biol Chem 263, 10812-6. Flores, O., Maldonado, E., and Reinberg, D. (1989). Factors involved in specific transcription by mammalian RNA polymerase H. Factors HE and HF independently interact with RNA polymerase H. J Biol Chem 264, 8913-21. 195 Fondell, J. D., Ge, H., and Roeder, R. G. (1996). Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex. Proc Natl Acad Sci U S A 93, 8329-33. Forget, D., Robert, F., Grondin, 6., Burton, Z. F ., Greenblatt, J ., and Coulombe, B. (1997). RAP74 induces promoter contacts by RNA polymerase H upstream and downstream of a DNA bend centered on the TATA box. Proc Natl Acad Sci U S A 94, 7150-7155. Frank, D. J., Tyree, C. M., George, C. P., and Kadonaga, J. T. (1995). Structure and fimction of the small subtmit of TFIIF (RAP30) fi'om Drosophila melanogaster. J Biol Chem 270, 6292-7. Franklin, C. C., McCulloch, A. V., and Kraft, A. S. (1995). In vitro association between the Jun protein family and the general transcription factors, TBP and TFHB. Biochem J 305, 967-74. Friedberg, E. C. (1996). Relationships between DNA repair and transcription. Annu Rev Biochem 65, 15-42. Fujinaga, K., Cujec, '1‘. P., Peng, J ., Garriga, J., Price, D. H., Grana, X., and Peterlin, B. M. (1998). The ability of positive transcription elongation factor B to transactivate human immunodeficiency virus transcription depends on a functional kinase domain, cyclin T1, and Tat. J Virol 72, 7154-7159. Garcia-Martinez, L. F., Mavankal, G., Neveu, J. M., Lane, W. S., Ivanov, D., and Gaynor, R. B. (1997). Pruification of a Tat-associated kinase reveals a TFIIH complex that modulates HIV-1 transcription. Embo J 16, 2836-2850. Garrett, K. P., Serizawa, H., Hanley, J. P., Bradsher, J. N., Tsuboi, A., Arai, N., Yokota, T., Arai, K., Conaway, R. C., and Conaway, J. W. (1992). The carboxyl terminus of RAP30 is similar in sequence to region 4 of bacterial sigma factors and is required for ftmction. J Biol Chem 26 7, 23942-23949. Ge, H., and Roeder, R. G. (1994a). The high mobility group protein HMGl can reversibly inhibit class H gene transcription by interaction with the TATA-binding protein. J Biol Chem 269, 17136-40. Ge, H., and Roeder, R. G. (1994b). Purification, cloning, and characterization ofa human coactivator, PC4, that mediates transcriptional activation of class H genes. Cell 78, 513- 23. Geiger, J. H., Hahn, S., Lee, S., and Sigler, P. B. (1996). Crystal structure of the yeast TFHA/TBP/DNA complex. Science 272, 830-6. 196 Gerard, M., Fischer, L., Moncollin, V., Chipoulet, J. M., Chambon, P., and Egly, J. M. (1991). Pmification and interaction properties of the human RNA polymerase B01) general transcription factor BTF2. J Biol Chem 266, 20940-5. Gibson, T. J ., Thompson, J. D., Blocker, A., and Kouzarides, T. (1994). Evidence for a protein domain superfamily shared by the cyclins, TFHB and RB/p107. Nucleic Acids Res 22, 946-52. Gill, G., Pascal, E., Tseng, Z. H., and Tjian, R. (1994). A glutamine-rich hydrophobic patch in transcription factor Spl contacts the dTAFHl 10 component of the Drosophila TFHD complex and mediates transcriptional activation. Proc Natl Acad Sci U S A 91 , 192-6. ' Gong, D. W., Hashimoto, S. ,Wada, K, Roeder, R. G. Nakatani, Y, and Horikoshi, M. (1992). Imperfect conservation of a sigma factor-like subregion in Xenopus general transcription factor RAP30. Nucleic Acids Res 20, 6414. Gong, D. W., Mortin, M. A., Horikoshi, M., and Nakatani, Y. (1995). Molecular cloning of cDNA encoding the small sublmit of Drosophila transcription initiation factor TFIIF. Nucleic Acids Res 23, 1882-6. Goodrich, J. A., Hoey, T., Thut, c. J., Admon, A., and Tjian, R. (1993). Drosophila TAFH40 interacts with both a VP16 activation domain and the basal transcription factor TFHB. Cell 75, 519-30. Goodrich, J. A., and Tjian, R. (1994a). TBP-TAP complexes: selectivity factors for eukaryotic transcription. Curr Opin Cell Biol 6, 403-9. Goodrich, J. A., and Tjian, R. (1994b). Transcription factors HE and HH and ATP hydrolysis direct promoter clearance by RNA polymerase H. Cell 7 7, 145-56. Grant, P. A., Duggan, L., Cote, J., Roberts, S. M., Brownell, J. E., Candau, R., Ohba, R., Owen-Hughes, T., Allis, C. D., Winston, F., Berger, S. L., and Workman, J. L. (1997). Yeast Gcn5 functions in two multisubtmit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. Genes Dev 11, 1640-1650. Grant, P. A., Schieltz, D., Pray-Grant, M. G., Steger, D. J ., Reese, J. C., Yates, J. R., 3rd, and Workman, J. L. (1998). A subset of TAF(H)s are integral components of the SAGA complex required for nucleosome acetylation and transcriptional stimulation. Cell 94, 45- 53. ' Greenleaf, A. L. (1993). Positive patches and negative noodles: linking RNA processing to transcription? Trends Biochem Sci 18, 117-9. 197 Groft, C. M., Uljon, S. N., Wang, R, and Werner, M. H. (1998). Structural homology between the Rap30 DNA-binding domain and linker histone H5: implications for preinitiation complex assembly. Proc Natl Acad Sci U S A 95, 9117-9122. Gu, W., and Roeder, R. G. (1997). Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595-606. Guermah, M., Malik, S., and Roeder, R. G. (1998). Involvement of TFHD and USA components in transcriptional activation of the human immunodeficiency virus promoter by NF-kappaB and Spl . Mol Cell Biol 18, 3234-44. Guzder, S. N., Qiu, H., Sommers, C. H., Sung, P., Prakash, L., and Prakash, S. (1994a). DNA repair gene RAD3 of S. cerevisiae is essential for transcription by RNA polymerase H. Nature 367, 91-94. Guzder, S. N., Sung, P., Bailly, V., Prakash, L., and Prakash, S. (1994b). RAD25 is a DNA helicase required for DNA repair and RNA polymerase H transcription. Nature 369, 578-581. Ha, 1., Lane, W. S., and Reinberg, D. (1991). Cloning of a human gene encoding the general transcription initiation factor HB. Nature 352, 689-95. Ha, L, Roberts, 8., Maldonado, E., Sun, X., Kim, L. U., Green, M., and Reinberg, D. (1993). Multiple functional domains of human transcription factor HB: distinct interactions with two general transcription factors and RNA polymerase H. Genes Dev 7, 1021-32. Hampsey, M. (1998). Molecular genetics of the RNA polymerase H general transcriptional machinery. Microbiol Mol Biol Rev 62, 465-503. Hampsey, M. (1997). A SAGA of histone acetylation and gene expression. Trends Genet 13, 427-429. Hanna Rose, W., and Hansen, U. (1996). Active repression mechanisms of eukaryotic transcription repressors. Trends Genet 12, 229-34. Hansen, S. K, Takada, S., Jacobson, R. H., Lis, J. T., and Tjian, R. (1997). Transcription properties of a cell type-specific TATA-binding protein, TRF. Cell 91 , 71-83. Hansen, S. K, and Tjian, R. (1995). TAFs and TFHA mediate difi‘erential iitilization of the tandem Adh promoters. Cell 82, 565-75. Harrison, S. C. (1991). A structmal taxonomy of DNA-binding domains. Nature 353, 7 15-7 19. 198 Hassig, C. A., Fleischer, T. C., Billin, A. N., Schreiber, S. L., and Ayer, D. E. (1997). Histone deacetylase activity is required for full transcriptional repression by mSin3A. Cell 89, 341-347. Hassig, C. A., Tong, J. K, Fleischer, T. C., Owa, T., Grable, P. G., Ayer, D. E., and Schreiber, S. L. (1998). A role for histone deacetylase activity in HDACl-mediated transcriptional repression. Proc Natl Acad Sci U S A 95, 3519-3524. Hawley, D. K, and Roeder, R. G. (1985). Separation and partial characterization of three functional steps in transcription initiation by human RNA polymerase H. J Biol Chem 260, 8163-72. Hayashi, P., Ishima, R., Liu, D., Tong, K. 1., Kim, S., Reinberg, D., Bagby, S., and Ikura, M. (1998). Human general transcription factor TFHB: conformational variability and interaction with VP16 activation domain. Biochemistry 37, 7941-7951. ' Heinzel, T., Lavinsky, R. M., Mullen, T. M., Soderstrom, M., Laherty, C. D., Torchia, J ., Yang, W. M., Brard, G., Ngo, S. D., Davie, J. R., Seto, E., Eisenman, R. N., Rose, D. W., Glass, C. K, and Rosenfeld, M. G. (1997). A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387, 43-48. Hekmatpanah, D. S., and Young, R. A. (1991). Mutations in a conserved region of RNA polymerase H influence the accuracy of mRNA start site selection. Mol Cell Biol 11, 5781-91. Hengartner, C. J., Myer, V. E., Liao, S. M., Wilson, C. J., Koh, S. S., and Young, R. A. (1998). Temporal regulation of RNA polymerase H by Srb10 and Kin28 cyclin- dependent kinases. Mol Cell 2, 43-53. Hengartner, C. J., Thompson, C. M., Zhang, J., Chao, D. M., Liao, S. M., Koleske, A. J ., Okamura, S., and Young, R. A. (1995). Association of an activator with an RNA polymerase H holoenzyme. Genes Dev 9, 897-910. Henry, N. L., Campbell, A. M., Feaver, W. J ., Poon, D., Weil, P. A., and Kornberg, R. D. (1994). TFHF-TAF-RNA polymerase H connection. Genes Dev 8, 2868-78. Henry, N. L., Sayre, M. H., and Kornberg, R. D. (1992). Purification and characterization of yeast RNA polymerase H general initiation factor g. J Biol Chem 26 7, 23388-92. Hernandez, N. (1993). TBP, a universal eukaryotic transcription factor? Genes Dev 7, 1291-308. . Hodo, H. G. d., and Blatti, S. P. (1977). Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA- dependent RNA polymerase H. Biochemistry 16, 2334-2343. 199 Hoey, T., Dynlacht, B. D., Peterson, M. G., Pugh, B. F., and Tjian, R. (1990). Isolation and characterization of the Drosophila gene encoding the TATA box binding protein, T'FHD. Cell 61, 1179-86. Hoffinann, A., Chiang, C. M., Oelgeschlager, T., Xie, X., Burley, S. K, Nakatani, Y., and Roeder, R. G. (1996). A histone octamer-like structure within TFHD. Nature 380, 356-359. Homnann, A., Oelgeschlager, T., and Roeder, R. G. (1997). Considerations of transcriptional control mechanisms: do TFHD-core promoter complexes recapitulate nucleosome-like firnctions? Proc Natl Acad Sci U S A 94, 8928-8935. Holstege, F. C., Fiedler, U., and Timmers, H. T. (1997). Three transitions in the RNA polymerase H transcription complex during initiation. Embo J 16, 7468-7480. Holstege, F. C., Tantin, D., Carey, M., van der Vliet, P. C., and Timmers, H. T. (1995). The requirement for the basal transcription factor HE is determined by the helical stability of promoter DNA. EMBO J 14, 810-9. Holstege, F. C., van der Vliet, P. C., and T immers, H. T. (1996). Opening of an RNA polymerase H promoter occms in two distinct steps and requires the basal transcription factors HE and IIH. EMBO J 15, 1666-77. Hoopes, B. C., LeBlanc, J. F., and Hawley, D. K (1992). Kinetic analysis of yeast TFHD-TATA box complex formation suggests a multi-step pathway. J Biol Chem 26 7, 1 1539-47. Horikoshi, M., Yamamoto, T., Ohkuma, Y., Weil, P. A., and Roeder, R. G. (1990). Analysis of structure-function relationships of yeast TATA box binding factor TFIID. Cell 61, 1171-8. Horiuchi, J ., Silverman, N., Marcus, G. A., and Guarente, L. (1995). ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCN 5 in a trimeric complex. Mol Cell Biol 15, 1203-1209. Horiuchi, J., Silverman, N. ,Pina, B., Marcus, G. A, and Guarente, L. (1997). ADAl, a novel component of the ADA/GCN 5 complex, has broader effects than GCN5, ADA2, or ADA3. Mol Cell Biol 17, 3220-3228. Hull, M. W., McKune, K, and Woychik, N. A. (1995). RNA polymerase H subunit RPB9 is required for accurate start site selection. Genes Dev 9, 481-90. Imbalzano, A. N., Kwon, H., Green, M. R., and Kingston, R. E. (1994). Facilitated binding of TATA-binding protein to nucleosomal DNA. Nature 3 70, 481-5. 200 Imbalzano, A. N., Zaret, K S., and Kingston, R. E. (1994). Transcription factor (TF) HB and TFHA can independently increase the affinity of the TATA-binding protein for DNA. J Biol Chem 269, 8280-6. Ingles, C. J ., Shales, M., Cress, W. D., Triezenberg, S. J., and Greenblatt, J. (1991). Reduced binding of TFHD to transcriptionally compromised mutants of VP16. Nature 351, 588-90. Inostroza, J ., Flores, O., and Reinberg, D. (1991). Factors involved in specific transcription by mammalian RNA polymerase H. Pmification and fimctional analysis of general transcription factor HE. J Biol Chem 266, 9304—8. Inostroza, J. A., Mermelstein, F. H., Ha, 1., Lane, W. S., and Reinberg, D. (1992). Drl, a TATA-binding protein-associated phosphoprotein and inhibitor of class H gene transcription. Cell 70, 477-89. Izban, M. G., and Luse, D. S. (1992a). Factor-stimulated RNA polymerase H transcribes at physiological elongation rates on naked DNA but very poorly on chromatin templates. J Biol Chem 267, 13647-55. Izban, M. G., and Luse, D. S. (1993a). The increment of SH-facilitated transcript cleavage varies dramatically between elongation competent and incompetent RNA polymerase H ternary complexes. J Biol Chem 268, 12874-85. Izban, M. G., and Luse, D. S. (1992b). The RNA polymerase H ternary complex cleaves the nascent transcript in a 3'--5' direction in the presence of elongation factor SH. Genes Dev 6, 1342-56. Izban, M. G., and Luse, D. S. (1993b). SH-facilitated transcript cleavage in RNA polymerase H complexes stalled early after initiation occms in primarily dinucleotide increments. J Biol Chem 268, 12864-73. Jacq, X., Brou, C., Lutz, Y., Davidson, 1., Chambon, P., and Tora, L. (1994). Human TAFH30 is present in a distinct TFHD complex and is required for transcriptional activation by the estrogen receptor. Cell 79, 107-17. Jeffrey, P. D., Russo, A. A., Polyak, K, Gibbs, E., Hurwitz, J ., Massague, J., and Pavletich, N. P. (1995). Mechanism of CDK activation revealed by the structm'e of a cyclinA-CDK2 complex. Nature 376, 313-320. Jeon, C., and Agarwal, K (1996). Fidelity of RNA polymerase H transcription controlled by elongation factor TFHS. Proc Natl Acad Sci U S A 93, 13677-13682. Jiang, Y., Smale, S. T., and Gralla, J. D. (1993). A common ATP requirement for open complex formation and transcription at promoters containing initiator or TATA elements. J Biol Chem 268, 6535-6540. 201 Jiang, Y., Triezenberg, S. J ., and Gralla, J. D. (1994). Defective transcriptional activation by diverse VP16 mutants associated with a common inability to form open promoter complexes. J Biol Chem 269, 5505-5508. Jiang, Y., Yan, M., and Gralla, J. D. (1995). Abortive initiation and first bond formation at an activated adenovirus E4 promoter. J Biol Chem 270, 27332-27338. Jiang, Y., Yan, M., and Gralla, J. D. (1996). A three-step pathway of transcription initiation leading to promoter clearance at an activation RNA polymerase H promoter. M01 Cell Biol 16, 1614-1621. Johnson, T. L., and Chamberlin, M. J. (1994). Complexes of yeast RNA polymerase II and RNA are substrates for T‘FHS- induced RNA cleavage. Cell 7 7, 217-224. Joliot, V., Demma, M., and Prywes, R. (1995). Interaction with RAP74 subunit of TFHF is required for transcriptional activation by serum response factor. Nature 373, 632-5. Jones, K A., and Peterlin, B. M. (1994). Control of RNA initiation and elongation at the I-HV-l promoter. Annu Rev Biochem 63, 717-743. Kaiser, K, and Meisteremst, M. (1996). The human general co-factors. Trends Biochem Sci 21, 342-345. Kaldis, P., Russo, A. A., Chou, H. S., Pavletich, N. P., and Solomon, M. J. (1998). Human and Yeast Cdk-activating Kinases (CAKs) Display Distinct Substrate Specificities. Mol Biol Cell 9, 2545-2560. Kaldis, P., Sutton, A., and Solomon, M. J. (1996). The Cdk-activating kinase (CAK) fiom budding yeast. Cell 86, 553-64. Kamakaka, R. T., Bulger, M., and Kadonaga, J. T. (1993). Potentiation of RNA polymerase H transcription by Gal4-VP16 during but not after DNA replication and chromatin assembly. Genes Dev 7, 1779-95. Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S. C., Heyman, R. A., Rose, D. W., Glass, C. K, and Rosenfeld, M. G. (1996). A CBP integrator complex mediates transcriptional activation and AP-l inhibition by nuclear receptors. Cell 85, 403-414. Kassavetis, G. A., Joazeiro, C. A., Pisano, M., Geiduschek, E. P., Colbert, T., Hahn, S., and Blanco, J. A. (1992). The role of the TATA-binding protein in the assembly and fimction of the multisubunit yeast RNA polymerase HI transcription factor, TFHIB. Cell 71, 1055-64. 202 Kasten, M. M., Dorland, S., and Stillman, D. J. (1997). A large protein complex containing the yeast Sin3p and de3p transcriptional regulators. Mol Cell Biol 17, 4852- 4858. Kaufinann, J., Ahrens, K, Koop, R., Smale, S. T., and Muller, R. (1998). C1F150, a human cofactor for transcription factor HD-dependent initiator function. Mol Cell Biol 18, 233-239. Kaufinann, J ., and Smale, S. T. (1994). Direct recognition of initiator elements by a component of the transcription factor HD complex. Genes Dev 8, 821-9. Kaufmann, J., Verrijzer, C. P., Shao, J., and Smale, S. T. (1996). CIF, an essential cofactor for TFIID-dependent initiator function. Genes Dev 10, 873-86. Kelleher, R. J. d., Flanagan, P. M., and Kornberg, R. D. (1990). A novel mediator between activator proteins and the RNA polymerase H transcription apparatus. Cell 61 , 1209-15. Kephart, D. D., Price, M. P., Burton, Z. P., Finkelstein, A., Greenblatt, J., and Price, D. H. (1993). Cloning of a Drosophila cDNA with sequence similarity to human transcription factor RAP74. Nucleic Acids Res 21 , 1319. Kephart, D. D., Wang, B. Q., Burton, Z. P., and Price, D. H. (1994). Functional analysis of Drosophila factor 5 (TFHF), a general transcription factor. J Biol Chem 269, 13536- 43. Killeen, M., Coulombe, B., and Greenblatt, J. (1992). Recombinant TBP, transcription factor HB, and RAP30 are suficient for promoter recognition by mammalian RNA ' polymerase H. J Biol Chem 26 7, 9463-6. Kim, J. L., Nikolov, D. B., and Burley, S. K (1993). Co-crystal structure of TBP recognizing the minor groove of a TATA element. Natme 365, 520-527. Kim, T. K, Lagrange, T., Wang, Y. H., Griflith, J. D., Reinberg, D., and Ebright, R. H. (1997). Trajectory of DNA in the RNA polymerase H transcription preinitiation complex. Proc Natl Acad Sci U S A 94, 12268-12273. Kim, T. K, and Roeder, R. G. (1994). Proline-rich activator CTF 1 targets the TFHB assembly step during transcriptional activation. Proc Natl Acad Sci U S A 91 , 4170-4. Kim, U., Qin, X. F., Gong, 8., Stevens, 8., Luo, Y., Nussenzweig, M., and Roeder, R. G. (1996). The B-cell-specific transcription coactivator OCA-B/OBF-l/Bob-l is essential for normal production of immunoglobulin isotypes. Nature 383, 542-7. Kim, Y., Geiger, J. H., Hahn, S., and Sigler, P. B. (1993). Crystal structure of a yeast TBP/TATA-box complex. Nature 365, 512-520. 203 Kim, Y. J ., Bjorkllmd, S., Li, Y., Sayre, M. H., and Kornberg, R. D. (1994). A multiprOtein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase H. Cell 7 7, 599-608. Kingston, R. E., Bunker, C. A., and Imbalzano, A. N. (1996). Repression and activation by multiprotein complexes that alter chromatin structure. Genes Dev 10, 905-20. Kitajima, S., Chibazakura, T., Yonaha, M., and Yasukochi, Y. (1994). Regulation of the human general transcription initiation factor TFHF by phosphorylation. J Biol Chem 269, 29970-7. Kitajima, S., Tanaka, Y., Kawaguchi, T., Nagaoka, T., Weissman, S. M., and Yasukochi, Y. (1990). A heteromeric transcription factor required for mammalian RNA polymerase H. Nucleic Acids Res 18, 4843-4849. Klages, N., and Strubin, M. (1995). Stimulation of RNA polymerase H transcription initiation by recruitment of TBP in vivo. Nature 374, 822-3. Klein, C., and Struhl, K (1994). Increased recruitment of TATA-binding protein to the promoter by transcriptional activation domains in vivo. Science 266, 280-282. K1emm,R. D., Goodrich J. A., Zhou, s., and Tjian, R. (1995). Molecular cloning and expression of the 32-kDa subunit of human TFHD reveals interactions with VP16 and TFHB that mediate transcriptional activation. Proc Natl Acad Sci U S A 92, 5788-92. Kobayashi, N., Boyer, T. G., and Berk, A. J. (1995). A class of activation domains interacts directly with TFHA and stimulates TFHA-TFHD-promoter complex assembly. Mol Cell Biol 15, 6465-73. Kobayashi, N., Horn, P. J ., Sullivan, S. M., Triezenberg, S. J ., Boyer, T. G., and Berk, A. J. (1998). DA-complex assembly activity required for VP 1 6C transcriptional activation. Mol Cell Biol 18, 4023-4031. Koh, S. S., Ansari, A. Z., Ptashne, M., and Young, R. A. (1998). An activator target in the RNA polymerase H holoenzyme. M01 Cell 1, 895-904. Kokubo, T., Swanson, M. J ., Nishikawa, J. 1., Hinnebusch, A: G., and Nakatani, Y. (1998). The yeast TAF145 inhibitory domain and TFHA competitively bind to TATA- binding protein. Mol Cell Biol 18, 1003-1012. Kokubo, T., Takada, R., Yamashita, S., Gong, D. W., Roeder, R. G., Horikoshi, M., and Nakatani, Y. (1993). Identification of TFHD components required for transcriptional activation by upstream stimulatory factor. J Biol Chem 268, 17 554-8. 204 Kokubo, T., Yamashita, S., Horikoshi, M., Roeder, R. G., and Nakatani, Y. (1994). Interaction between the N-terminal domain of the 230-kDa subunit and the TATA box- binding subunit of TFHD negatively regulates TATA-box binding. Proc Natl Acad Sci U S A 91 , 3520-4. Koleske, A. J ., Buratowski, S., Nonet, M., and Young, R. A. (1992). A novel transcription factor reveals a fimctional link between the RNA polymerase H CTD and TFHD. Cell 69, 883-94. Koleske, A. J ., and Young, R. A. (1995). The RNA polymerase H holoenzyme and its implications for gene regulation. Trends Biochem Sci 20, 113-6. Koleske, A. J ., and Young, R. A. (1994). An RNA polymerase H holoenzyme responsive to activators. Natm'e 368, 466-9. Kolodziej, P., and Young, R. A. (1989). RNA polymerase H subunit RPB3 is an essential component of the mRNA transcription apparatus. Mol Cell Biol 9, 5387-94. Kolodziej, P. A., Woychik, N., Liao, S. M., and Young, R. A. (1990). RNA polymerase H subunit composition, stoichiometry, and phosphorylation. Mol Cell Biol 10, 1915-20. Kolodziej, P. A., and Young, R. A. (1991). Mutations in the three largest subunits of yeast RNA polymerase H that afl‘ect enzyme assembly. Mol Cell Biol 11, 4669-78. Kraus, W. L., and Kadonaga, J. T. (1998). p300 and estrogen receptor cooperatively activate transcription via difl‘erential enhancement of initiation and reinitiation. Genes Dev 12, 331-342. Kretzschmar, M., Meisteremst, M., and Roeder, R. G. (1993). Identification of human DNA topoisomerase I as a cofactor for activator- dependent transcription by RNA polymerase H. Proc Natl Acad Sci U S A 90, 11508-11512. Kretzschmar, M., Stelzer, G., Roeder, R. G., and Meisteremst, M. (1994). RNA polymerase H cofactor PC2 facilitates activation of transcription by GAL4-AH in vitro. Mol Cell Biol 14, 3927-37. Krumm, A., Hickey, L. B., and Groudine, M. (1995). Promoter-proximal pausing of RNA polymerase H defines a general rate- limiting step after transcription initiation. Genes Dev 9, 559-572. Kuldell, N. H., and Buratowski, S. (1997). Genetic analysis of the large subunit of yeast transcription factor HE reveals two regions with distinct fimctions. Mol Cell Biol 1 7, 5288-5298. 205 Kwok, R. P., Lundblad, J. R., Chrivia, J. C., Richards, J. P., Bachinger, H. P., Brennan, R. G., Roberts, S. G., Green, M. R., and Goodman, R. H. (1994). Nuclear protein CBP is a coactivator for the transcription factor CREB. Natm'e 370, 223-226. Kwon, H., Imbalzano, A. N., Khavari, P. A., Kingston, R. E., and Green, M. R. (1994). Nucleosome disruption and enhancement of activator binding by a human SWl/SNF complex. Nature 370, 477-81. Lagrange, T., Kapanidis, A. N., Tang, H., Reinberg, D., and Ebright, R. H. (1998). New core promoter element in RNA polymerase H-dependent transcription: sequence-specific DNA binding by transcription factor HB. Genes Dev 12, 34-44. Lagrange, T., Kim, T. K, Orphanides, G., Ebright, Y. W., Ebright, R. H., and Reinberg, D. (1996). High-resolution mapping of nucleoprotein complexes by site-specific protein- DNA photocrosslinking: organization of the human TBP-TFHA- TFHB-DNA quaternary complex. Proc Natl Acad Sci U S A 93, 10620-10625. Laherty, C. D., Yang, W. M., Sun, J. M., Davie, J. R., Seto, E., and Eisenman, R. N. (1997). Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression. Cell 89, 349-356. Landick, R. (1997). RNA polymerase slides home: pause and termination site recognition. Cell 88, 741-744. Laybourn, P. J., and Dahmus, M. E. (1989). Transcription-dependent structural changes in the C-terminal domain of mammalian RNA polymerase subunit Ha/o. J Biol Chem 264, 6693-6698. Lee, D., and Lis, J. T. (1998). Transcriptional activation independent of TFHH kinase and ' the RNA polymerase H mediator in vivo. Nature 393, 389-392. Lee, H., Kraus, K W., Wolfirer, M. F., and Lis, J. T. (1992). DNA sequence requirements for generating paused polymerase at the start of hsp70. Genes Dev 6, 284-95. Lee, J. M., and Greenleaf, A. L. (1991). CTD kinase large subunit is encoded by CTKI, a gene required for normal growth of Saccharomyces cerevisiae. Gene Expr 1, 149-67. Lee, S., and Hahn, S. (1995). Model for binding of transcription factor TFHB to the TBP- DNA complex. Nature 376, 609-12. Lei, L., Ren, D., Finkelstein, A., and Bmton, Z. F. (1998). Functions of the N- and C- terminal domains of human RAP74 in transcriptional initiation, elongation, and recycling of RNA polymerase H. Mol Cell Biol 18, 2130-42. 206 Leong, G. M., Wang, K S., Marton, M. J., Blanco, J. C., Wang, 1. M., Rolfes, R. J ., Ozato, K, and Segars, J. H. (1998). Interaction between the retinoid X receptor and _ transcription factor HB is ligand-dependent in vivo. J Biol Chem 273, 2296-2305. Leuther, K K, Bushnell, D. A., and Kornberg, R. D. (1996). Two-dimensional crystallography of TFIIB- and HE-RNA polymerase H complexes: implications for start site selection and initiation complex formation. Cell 85 , 77 3-9. Li, Y., Bjorkltmd, S., Kim, Y. J ., and Kornberg, R. D. (1996). Yeast RNA polymerase H holoenzyme. Methods Enzym01273, 172-5. Li, Y., Flanagan, P. M., Tschochner, H., and Kornberg, R. D. (1994). RNA polymerase II initiation factor interactions and transcription start site selection. Science 263, 805-7. Liao, S. M., Zhang, J., Jeffery, D. A., Koleske, A. J., Thompson, C. M., Chao, D. M., Viljoen, M., van Vuuren, H. J., and Young, R. A. (1995). A kinase-cyclin pair in the RNA polymerase H holoenzyme [see comments]. Nature 374, 193-6. Lin, Y. 8., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R. (1991). Binding of general transcription factor TFHB to an acidic activating region. Nature 353, 569-71. Linn, S. 'C., and Luse, D. S.,(1991). RNA polymerase H elongation complexes paused after the synthesis of 15- or 35-base transcripts have different structures. Mol Cell Biol 11, 1508-22. Lis, J ., and Wu, C. (1993). Protein traffic on the heat shock promoter: parking, stalling, and trucking along. Cell 74, 1-4. Liu, D., lshima, R., Tong, K 1., Bagby, S., Kokubo, T., Muhandirarn, D. R., Kay, L. E., Nakatani, Y., and lkura, M. (1998). Solution structure of a TBP-TAF(H)230 complex: protein mimicry of the minor groove surface of the TATA box unwound by TBP. Cell 94, 573-583. Liu, M., Xie, Z., and Price, D. H. (1998). A Human RNA Polymerase H Transcription Termination Factor Is a SWIZ/SNFZ Family Member. J Biol Chem 273, 25541-25544. Liu, X., Miller, C. W., Koeffler, P. H., and Berk, A. J. (1993). The p53 activation domain binds the TATA box-binding polypeptide in Holo-TFHD, and a neighboring p53 domain inhibits transcription. Mol Cell Biol 13, 3291-300. Lobo, S.’ M., Tanaka, M., Sullivan, M. L., and Hernandez, N. (1992). A TBP complex essential for transcription from TATA-less but not TATA-containing RNA polymerase HI promoters is part of the TFHIB fiaction. Cell 71 , 1029-40. 207 Lofquist, A. K, Li, H., Imboden, M. A., and Paule, M. R. (1993). Promoter opening (melting) and transcription initiation by RNA polymerase 1 requires neither nucleotide beta,gamma hydrolysis nor protein phosphorylation. Nucleic Acids Res 21 , 3233-3238. Lorch, Y., LaPointe, J. W., and Kornberg, R. D. (1992). Initiation on chromatin templates in a yeast RNA polymerase H transcription system. Genes Dev 6, 2282-7. Lu, H., Fisher, R. P., Bailey, P., and Levine, A. J. (1997). The CDK7-cycH-p36 complex of transcription factor HH phosphorylates p53, enhancing its sequence-specific DNA binding activity in vitro. Mol Cell Biol 1 7, 5923-5934. Lu, H., Flores, O., Weinmann, R., and Reinberg, D. (1991). The nonphosphorylated form of RNA polymerase H preferentially associates with the preinitiation complex. Proc Natl Acad Sci U S A 88, 10004-8. Lu, H., Zawel, L., Fisher, L., Egly, J. M., and Reinberg, D. (1992). Human general transcription factor IIH phosphorylates the C-terminal domain of RNA polymerase H. Name 358, 641-645. Luse, D. S., and Jacob, G. A. (1987). Abortive initiation by RNA polymerase H in vitro at the adenovirus 2 major late promoter. J Biol Chem 262, 14990-7. Ma, D., Olave, 1., Merino, A., and Reinberg, D. (1996). Separation of the transcriptional coactivator and antirepression ftmctions of transcription factor HA. Proc Natl Acad Sci U S A 93, 6583-8. Ma, D., Watanabe, H., Mermelstein, F., Admon, A., Oguri, K, Sun, X., Wada, T., Imai, T., Shiroya, T., Reinberg, D., and et a1. (1993). Isolation of a cDNA encoding the largest subunit of TFHA reveals functions important for activated transcription. Genes Dev 7, 2246-57. Madison, J. M., and Winston, F. (1997). Evidence that Spt3 functionally interacts With Motl , TFHA, and TATA- binding protein to confer promoter-specific transcriptional control in Saccharomyces cerevisiae. Mol Cell Biol 17, 287-295. Majello, B. ,Napolitano, G., De Luca, P., and Lania, L. (1998). Recruitment of human TBP selectively activates RNA polymerase H TATA- dependent promoters. J Biol Chem 273, 16509-16516. Makela, T. P., Parvin, J. D., Kim, J ., Huber, L. J., Sharp, P. A., and Weinberg, R. A. (1995). A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription. Proc Natl Acad Sci U S A 92, 5174-8. Maldonado, E., Drapkin, R., and Reinberg, D. (1996a). Purification of human RNA polymerase H and general transcription factors. Methods Enzym01274, 72-100. 208 Maldonado, E., Ha, 1., Cortes, P., Weis, L., and Reinberg, D. (1990). Factors involved in specific transcription by mammalian RNA polymerase H: role of transcription factors HA, HD, and HB during formation of a transcription-competent complex. Mol Cell Biol 10, 6335-47. Maldonado, E., Shiekhattar, R., Sheldon, M., Cho, H., Drapkin, R., Rickert, P., Lees, B, Anderson, C. W., Linn, S., and Reinberg, D. (1996b). A human RNA polymerase H complex associated with SRB and DNA-repair proteins. Nature 381 , 86-9. Malik, S., Guermah, M., and Roeder, R. G. (1998). A dynamic model for PC4 coactivator function in RNA polymerase H transcription. Proc Natl Acad Sci U S A 95, 2192-7. Malik, S., Lee, D. K, and Roeder, R. G. (1993). Potential RNA polymerase II-induced interactions of transcription factor TFHB. Mol Cell Biol 13, 6253-9. Marcus, G. A., Horiuchi, J., Silverman, N., and Guarente, L. (1996). ADA5/SPT20 links the ADA and SPT genes, which are involved in yeast transcription. Mol Cell Biol 16, 3197-3205. Marcus, G. A., Silverman, N., Berger, S. L., Horiuchi, J ., and Guarente, L. (1994). Functional similarity and physical association between GCN5 and ADA2: putative transcriptional adaptors. Embo J 13, 4807-4815. Markovtsov, V., Mustaev, A., and Goldfarb, A. (1996). Protein-RNA interactions in the active center of transcription elongation complex. Proc Natl Acad Sci U S A 93, 3221-6. Marshall, N. F., Peng, J ., Xie, Z., and Price, D. H. (1996). Control of RNA polymerase H elongation potential by a novel carboxyl- terminal domain kinase. J Biol Chem 271 , 27 176-27 183. Marshall, N. F., and Price, D. H. (1992). Control of formation of two distinct classes of RNA polymerase H elongation complexes. Mol Cell Biol 12, 2078-2090. Marshall, N. F., and Price, D. H. (1995). Purification of P-TEFb, a transcription factor required for the transition into productive elongation. J Biol Chem 270, 12335-12338. Martinez, E., Chiang, C. M., Ge, H., and Roeder, R. G. (1994). TATA-binding protein- associated factor(s) in TFIID function through the initiator todirect basal transcription from a TATA-less class H promoter. EMBO J 13, 3115-26. Martinez, E., Ge, H., Tao, Y., Yuan, C. X., Palhan, V., and Roeder, R. G. (1998). Novel cofactors and TFHA mediate functional core promoter selectivity by the human TAFHlSO-containing TFHD complex. Mol Cell Biol 18, 6571-6583. 209 Martinez-Balbas, M. A., Bannister, A. J ., Martin, K, Haus-Seuffert, P., Meisteremst, M., and Kouzarides, T. (1998). The acetyltransferase activity of CBP stimulates transcription. Embo J 17, 2886-2893. Matsui, T., Segall, J ., Weil, P. A., and Roeder, R. G. (1980). Multiple factors required for accurate initiation of transcription by pmified RNA polymerase H. J Biol Chem 255, 1 1992-1 1996. Matsuzaki, H., Kassavetis, G. A., and Geiduschek, E. P. (1994). Analysis of RNA chain elongation and termination by Saccharomyces cerevisiae RNA polymerase IH. J Mol Biol 235, 1173-1192. Maxon, M. E., Goodrich, J. A., and Tjian, R. (1994). Transcription factor HE binds preferentially to RNA polymerase Ha and recruits TFIH-I: a model for promoter clearance. Genes Dev 8, 515-24. Maxon, M. E., and Tjian, R. (1994). Transcriptional activity of transcription factor HE is dependent on zinc binding. Proc Natl Acad Sci U S A 91 , 9529-33. Mazzarelli, J. M., Mengus, G., Davidson, 1., and Ricciardi, R. P. (1997). The transactivation domain of adenovirus ElA interacts with the C terminus of human TAF(H)135. J Virol 71, 7978-7983. McCracken, S., Fong, N., Rosonina, E., Yankulov, K, Brothers, G., Siderovski, D., Hessel, A., Foster, S., Shuman, S., and Bentley, D. L. (1997a). 5'-Capping enzymes are targeted to pro-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase H. Genes Dev 11, 3306-3318. McCracken, S., Fong, N., Yankulov, K, Ballantyne, 8., Pan, G., Greenblatt, J ., Patterson, S. D., Wickens, M., and Bentley, D. L. (1997b). The C-terminal domain of RNA polymerase H couples mRNA processing to transcription. Nature 385, 357-61. McCracken, S., and Greenblatt, J. (1991). Related RNA polymerase-binding regions in human RAP30/74 and Escherichia coli sigma 70. Science 253, 900-2. McEwan, I. J ., Dahlman-Wright, K, Ford, J ., and Wright, A. P. (1996). Functional interaction of the c-Myc transactivation domain with the TATA binding protein: evidence for an induced fit model of transactivation domain folding. Biochemistry 35, 9584-9593. McEwan, I. J ., and Gustafsson, J. (1997). Interaction of the human androgen receptor transactivation function with the general transcription factor TFHF. Proc Natl Acad Sci U S A 94, 8485-8490. McKune, K, Moore, P. A., Hull, M. W., and Woychik, N. A. (1995). Six human RNA polymerase sublmits flmctionally substitute for their yeast counterparts. Mol Cell Biol 15, 6895-6900. 210 Meisteremst, M., and Roeder, R. G. (1991). Family of proteins that interact with TFHD and regulate promoter activity. Cell 67, 557-67. Meisteremst, M., Roy, A. L., Lieu, H. M., and Roeder, R. G. (1991). Activation of class H gene transcription by regulatory factors is potentiated by a novel activity. Cell 66, 98 l- 93. Meisteremst, M., Stelzer, G., and Roeder, R. G. (1997). Poly(ADP-ribose) polymerase enhances activator-dependent transcription in vitro. Proc Natl Acad Sci U S A 94, 2261- 5. Melcher, K, and Johnston, S. A. (1995). GAL4 interacts with TATA-binding protein and coactivators. Mol Cell Biol 15, 2839-48. Merino, A., Madden, K R., Lane, W. S., Champoux, J. J., and Reinberg, D. (1993). DNA topoisomerase I is involved in both repression and activation of transcription. Nature 365 , 227-32. Mermelstein, F., Yeung, K, Cao, J ., Inostroza, J. A., Erdjument-Bromage, H., Eagelson, K, Landsman, D., Levitt, P., Tempst, P., and Reinberg, D. (1996). Requirement of a corepressor for Drl-mediated repression of transcription. Genes Dev 10, 1033-1048. Metzger, W., Schickor, P., and Hermann, H. (1989). A cinematographic view of Escherichia coli RNA polymerase translocation. Embo J 8, 2745-2754. Mitchell, P. J ., and Tjian, R (1989). Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245, 371-8. Mittal, V., and Hernandez, N. (1997). Role for the amino-terminal region of human TBP in U6 snRNA transcription. Science 275, 1136-1140. Mizzen,.C. A., Yang, X. J ., Kokubo, T., Brownell, J. E., Bannister, A. J ., Owen Hughes, T., Workman, J., Wang, L., Berger, S. L., Kouzarides, T., Nakatani, Y., and Allis, C. D. (1996). The TAF(H)250 sublmit of TFHD has histone acetyltransferase activity. Cell 87, 1261-70. Moqtaderi, Z., Bai, Y., Poon, D., Weil, P. A., and Struhl, K (1996). TBP-associated factors are not generally required for transcriptional activation in yeast. Nature 383, 188- 91. Mustaev, A., Kozlov, M., Markovtsov, V., Zaychikov, E., Denissova, L., and Goldfarb, A. (1997). Modular organization of the catalytic center of RNA polymerase. Proc Natl Acad Sci U S A 94, 6641-6645. 211 Nagy, L., Kao, H. Y., Chakravarti, D., Lin, R. J., Hassig, C. A., Ayer, D. E., Schreiber, S. L., and Evans, R. M. (1997). Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell 89, 373-380. Nakajima, N., Horikoshi, M., and Roeder, R. G. (1988). Factors involved in specific transcription by mammalian RNA polymerase H: purification, genetic specificity, and TATA box-promoter interactions of TFIID. Mol Cell Biol 8, 4028-40. Nakatani, Y., Bagby, S., and lkura, M. (1996). The histone folds in transcription factor TFHD. J Biol Chem 271, 6575-8. Nakatani, Y., Horikoshi, M., Brenner, M., Yamamoto, T., Besnard, F., Roeder, R. G., and Freese, E. (1990). A downstream initiation element required for efficient TATA box binding and in vitro function of TFHD. Nature 348, 86-8. Nikolov, D. B., Chen, H., Halay, E. D., Hoffinan, A., Roeder, R. G., and Burley, S. K (1996). Crystal structure of a human TATA box-binding protein/TATA element complex. Proc Natl Acad Sci U S A 93, 4862-7. Nikolov, D. B., Chen, H., Halay, E. D., Usheva, A. A., Hisatake, K, Lee, D. K, Roeder, R. G., and Burley, S. K (1995). Crystal structure of a TFHB-TBP-TATA-element ternary complex. Nature 3 7 7, 119-28. Nikolov, D. B., Hu, S. H., Lin, J ., Gasch, A., Hoffinann, A., Horikoshi, M., Chua, N. H., Roeder, R. G., and Burley, S. K (1992). Crystal structure of TFHD TATA-box binding protein. Nature 360, 40-6. Nishikawa, J ., Kokubo, T., Horikoshi, M., Roeder, R. G., and Nakatani, Y. (1997). Drosophila TAF(H)230 and the transcriptional activator VP 1 6 bind competitively to the TATA box-binding domain of the TATA box-binding protein. Proc Natl Acad Sci U S A 94, 85-90. Nonet, M. L., and Young, R. A. (1989). Intragenic and extragenic suppressors of mutations in the heptapeptide repeat domain of Saccharomyces cerevisiae RNA polymerase H. Genetics 123, 715-24. Nudler, E., Avetissova, E., Markovtsov, V., and Goldfarb, A. (1996). Transcription processivity: protein-DNA interactions holding together the elongation complex. Science 273, 21 1-7. Nudler, E., Goldfarb, A., and Kashlev, M. (1994). Discontinuous mechanism of transcription elongation. Science 265, 793-6. Nudler, E., Kashlev, M., Nikiforov, V., and Goldfarb, A. (1995). Coupling between transcription termination and RNA polymerase inchworming. Cell 81 , 351-7. 212 Nudler, E., Mustaev, A., Lukhtanov, E., and Goldfarb, A. (1997). The RNA-DNA hybrid maintains the register of transcription by preventing backtracking of RNA polymerase. Cell 89, 33-41. O. Brien, T., Hardin, S., Greenleaf, A., and Lis, J. T. (1994). Phosphorylation of RNA polymerase H C-terminal domain and transcriptional elongation. Nature 3 70, 7 5-7. O. Brien, T., and Lis, J. T. (1991). RNA polymerase H pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene. Mol Cell Biol 11, 5285-90. Oelgeschlager, T., Chiang, C. M., and Roeder, R. G. (1996). Topology and reorganization of a human TFHD-promoter complex. Nature 382, 735-8. Oelgeschlager, T., Tao, Y., Kang, Y. K, and Roeder, R. G. (1998). Transcription activation via enhanced preinitiation complex assembly in a human cell-fiee system lacking TAFHs. Mol Cell 1, 925-931. Ogryzko, V. V., Kotani, T., Zhang, X., Schlitz, R. L., Howard, T., Yang, X. J ., Howard, B. H., Qin, J ., and Nakatani, Y. (1998). Histone-like TAFs within the PCAF historic acetylase complex. Cell 94, 35-44. Ohkuma, Y., Hashimoto, S., Wang, C. K, Horikoshi, M., and Roeder, R. G. (1995). Analysis of the role of TFHE in basal transcription and TFHH-mediated carboxy-terminal domain phosphorylation through structure-fimction studies of TFHE-alpha. Mol Cell Biol 15, 4856-66. Ohkuma, Y., and Roeder, R. G. (1994). Regulation of TFHH ATPase and kinase activities by TFHE during active initiation complex formation. Name 368, 160-3. Ohkuma, Y., Sumimoto, H., Hofinann, A., Shimasaki, S., Horikoshi, M., and Roeder, R. G. (1991). Structural motifs and potential sigma homologies in the large subunit of human general transcription factor TFHE. Nature 354, 398-401. Ohkuma, Y., Sumimoto, H., Horikoshi, M., and Roeder, R. G. (1990). Factors involved in specific transcription by mammalian RNA polymerase H: pmification and characterization of general transcription factor TFHE. Proc Natl Acad Sci U S A 87, 9163-7. Orphanides, G., Lagrange, T., and Reinberg, D. (1996). The general transcription factors of RNA polymerase H. Genes Dev 10, 2657-83. Orphanides, G., LeRoy, G., Chang, C. H., Luse, D. S., and Reinberg, D. (1998). FACT, a factor that facilitates transcript elongation through nucleosomes. Cell 92, 105-116. 213 Ossipow, V., Tassan, J. P., Nigg, E. A., and Schibler, U. (1995). A mammalian RNA polymerase H holoenzyme containing all components required for promoter-specific transcription initiation. Cell 83, 137-46. Ouzounis, C., and Sander, C. (1992). TFHB, an evolutionary link between the transcription machineries of archaebacteria and eukaryotes. Cell 71, 189-90. Ozer, J ., Moore, P. A., Bolden, A. H., Lee, A., Rosen, C. A., and Lieberman, P. M. (1994). Molecular cloning of the small (gamma) sublmit of human TFHA reveals functions critical for activated transcription. Genes Dev 8, 2324-35. Pan, G., Aso, T., and Greenblatt, J. (1997). Interaction of elongation factors TFHS and elongin A with a human RNA polymerase H holoenzyme capable of promoter-specific initiation and responsive to transcriptional activators. J Biol Chem 2 72, 24563-24571. Pan, G., and Greenblatt, J. (1994). Initiation of transcription by RNA polymerase H is limited by melting of the promoter DNA in the region immediately upstream of the initiation site. J Biol Chem 269, 30101-4. Parada, C. A., and Roeder, R. G. (1996). Enhanced processivity of RNA polymerase H triggered by Tat-induced phosphorylation of its carboxy-terminal domain. Nature 384, 37 5-8. ' Paranjape, S. M., Kamakaka, R. T., and Kadonaga, J. T. (1994). Role of chromatin structm'e in the regulation of transcription by RNA polymerase H. Annu Rev Biochem 63, 265-97. Park, E., Guzder, S. N., Koken, M. H., Jaspers-Dekker, I., Weeda, G., Hoeijmakers, J. H., Prakash, S., and Prakash, L. (1992). RAD25 (SSL2), the yeast homolog of the human xeroderma pigrnentosmn group B DNA repair gene, is essential for viability. Proc Natl Acad Sci U S A 89, 11416-11420. Parvin, J. D., and Sharp, P. A. (1993). DNA topology and a minimal set of basal factors for transcription by RNA polymerase H. Cell 73, 533-40. Parvin, J. D., Shykind, B. M., Meyers, R. E., Kim, J ., and Sharp, P. A. (1994). Multiple sets of basal factors initiate transcription by RNA polymerase H. J Biol Chem 269, 18414-21. . Pause, A., Aso, T., Linehan, W. M., Conaway, J. W., Conaway, R. C., and Klausner, R. D. (1996). Interaction of von Hippel-Lindau tumor suppressor gene product with elongin. Methods Enzym01274, 436-441. Pazin, M. J ., and Kadonaga, J. T. (1997). What's up and down with histone deacetylation and transcription? Cell 89, 325-328. 214 Pearson, A., and Greenblatt, J. (1997). Modular organization of the E2Fl activation domain and its interaction with general transcription factors TBP and TFIIH. Oncogene 15, 2643-2658. Peng, J., Marshall, N. F., and Price, D. H. (1998a). Identification of a cyclin subunit required for the function of Drosophila P-TEFb. J Biol Chem 273, 13855-13860. Peng, J ., Zhu, Y., Milton, J. T., and Price, D. H. (1998b). Identification of multiple cyclin subunits of human P-TEFb. Genes Dev 12, 755-762. Peterson, M. G., Inostroza, J ., Maxon, M. E., Flores, O., Admon, A., Reinberg, D., and Tjian, R. (1991). Structure and functional properties of human general transcription factor HE. Nature 354, 369-73. Peterson, M. G., Tanese, N., Pugh, B. F., and Tjian, R. (1990). Functional domains and upstream activation properties of cloned human TATA binding protein. Science 248, 1625-1630. ' Pina, B., Berger, 8., Marcus, G. A., Silverman, N., Agapite, J., and Guarente, L. (1993). ADA3: a gene, identified by resistance to GAL4-VP16, with properties similar to and difl‘erent fiorn those of ADA2. Mol Cell Biol 13, 5981-5989. Pinto, 1., Ware, D. E., and Hampsey, M. (1992). The yeast SUA7 gene encodes a homolog of human transcription factor TFHB and is required for normal start site selection in vivo. Cell 68, 977-88. Pinto, 1., Wu, W. H., Na, J. G., and Hampsey, M. (1994). Characterization of sua7 mutations defines a domain of TFHB involved in transcription start site selection in yeast. J Biol Chem 269, 30569-73. Pollard, K J ., and Peterson, C. L. (1997). Role for ADA/GCN5 products in antagonizing chromatin-mediated transcriptional repression. Mol Cell Biol 1 7, 6212-6222. Polyakov, A., Severinova, E., and Darst, S. A. (1995). Three-dimensional structure of E. coli core" RNA polymerase: promoter binding and elongation conformations of the enzyme. Cell 83, 365-373. Powell, W., Bartholomew, B., and Reines, D. (1996). Elongation factor SH contacts the 3'-end of RNA in the RNA polymerase H elongation complex. J Biol Chem 271, 22301- 22304. Powell, W., and Reines, D. (1996). Mutations in the second largest subunit of RNA polymerase H cause 6- azamacil sensitivity in yeast and increased transcriptional arrest in vitro. J Biol Chem 271, 6866-6873. 215 Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1989). Dynamic interaction between a Drosophila transcription factor and RNA polymerase H. Mol Cell Biol 9, 1465-75. Pugh, B. F., and Tjian, R. (1990). Mechanism of transcriptional activation by Spl: evidence for coactivators. Cell 61 , 1 187-97. Pugh, B. F., and Tjian, R. (1991). Transcription from a TATA-less promoter requires a multisubunit TFHD complex. Genes Dev 5, 1935-45. Purnell, B. A., Emanuel, P. A., and Gilmour, D. S. (1994). TFIH) sequence recognition of the initiator and sequences farther downstream in Drosophila class H genes. Genes Dev 8, 830-42. Quinn, J., Fyrberg, A. M., Ganster, R. W., Schmidt, M. C., and Peterson, C. L. (1996). DNA-binding properties of the yeast SWIISNF complex. Nature 379, 844-847. Qureshi, S. A., Khoo, B., Baumann, P., and Jackson, S. P. (1995). Molecular cloning of the transcription factor TFHB homolog from Sulfolobus shibatae. Proc Natl Acad Sci U S A 92, 6077-81. Ranish, J. A., Lane, W. S., and Hahn, S. (1992). Isolation of two genes that encode subunits of the yeast transcription factor HA. Science 255, 1127-9. Rasmussen, E. B., and Lis, J. T. (1993). In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes. Proc Natl Acad Sci U S A 90, 7923-7. Reinberg, D., Horikoshi, M., and Roeder, R. G. (1987). Factors involved in specific transcription in mammalian RNA polymerase H. Functional analysis of initiation factors HA and HD and identification of a new factor operating at sequences downstream of the initiation site. J Biol Chem 262, 3322-30. Reinberg, D., and Roeder, R. G. (1987). Factors involved in specific transcription by mammalian RNA polymerase H. Transcription factor HS stimulates elongation of RNA chains. J Biol Chem 262, 3331-7. Reines, D., Chamberlin, M. J ., and Kane, C. M. (1989). Transcription elongation factor SH (TF HS) enables RNA polymerase H to elongate through a block to transcription in a human gene in vitro. J Biol Chem 264, 10799-10809. Reines, D., Conaway, J. W., and Conaway, R. C. (1996). The RNA polymerase H general elongation factors. Trends Biochem Sci 21 , 351-355. Robert, F., Douziech, M., Forget, D., Egly, J. M., Greenblatt, J ., Bmton, Z. P., and Coulombe, B. (1998). Wrapping of promoter DNA around the RNA polymerase H initiation complex induced by TFHF. Mol Cell 2, 341-51. 216 Robert, F., Forget, D., Li, J ., Greenblatt, J ., and Coulombe, B. (1996). Localization of subunits of transcription factors HE and HF immediately upstream of the transcriptional initiation site of the adenovirus major late promoter. J Biol Chem 271, 8517-20. Roberts, S. G., and Green, M. R. (1994). Activator-induced conformational change in general transcription factor TFHB. Nature 371, 717-20. Roberts, S. G., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R. (1993). Interaction between an acidic activator and transcription factor TFHB is required for transcriptional activation. Nature 363, 741-4. Roberts, S. M., and Winston, F. (1997). Essential fimctional interactions of SAGA, a Saccharomyces cerevisiae complex of Spt, Ada, and Gcn5 proteins, with the Snf/Swi and Stir/mediator complexes. Genetics 14 7, 451-465. Roberts, S. M., and Winston, F. (1996). SPT20/ADA5 encodes a novel protein ftmctionally related to the TATA- binding protein and important for transcription in Saccharomyces cerevisiae. Mol Cell Biol 16, 3206-3213. Rochette-Egly, 0., Adam, S., Rossignol, M., Egly, J. M., and Chambon, P. (1997). Stimulation of RAR alpha activation fimction AF-l through binding to the general transcription factor TFHH and phosphorylation by CDK7 . Cell 90, 97-107. Roe, J. H., Burgess, R. R., and Record, M. T., Jr. (1985). Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening. J Mol Biol 184, 441-53. Roeder, R. G. (1991). The complexities of eukaryotic transcription initiation: regulation of preinitiation complex assembly. Trends Biochem Sci 16, 402-8. Roeder, R. G. (1976). Eukaryotic nuclear RNA polymerases. pp. 285-329. In: Losick R, Chamberlin M, ed. RNA polymerase. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory 58, 1 1. Roeder, R. G. (1996). The role of general initiation factors in transcription by RNA polymerase H. Trends Biochem Sci 21 , 327-35. Rundlett, S. E., Carmen, A. A., Kobayashi, K, Bavykin, S., Turner, B. M., and Grunstein, M. (1996). I-IDAl and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription. Proc Natl Acad Sci U S A 93, 14503-14508. Ruppert, S., and Tjian, R. (1995). Human TAFH250 interacts with RAP74: implications for RNA polymerase H initiation. Genes Dev 9, 2747-55. 217 Sakurai, H., and Fukasawa, T. (1998). Flmctional correlation among Gall l, transcription factor (TF) HE, and TFIIH in Saccharomyces cerevisiae. Gall l and TFHE cooperatively enhance TFIIH-mediated phosphorylation of RNA polymerase H carboxyl- terminal domain sequences. J Biol Chem 273, 9534—9538. Sakurai, H., and Fukasawa, T. (1997). Yeast Gall l and transcription factor HE function through a common pathway in transcriptional regulation. J Biol Chem 272, 32663-32669. Sakurai, H., Kim, Y. J ., Ohishi, T., Kornberg, R. D., and Fukasawa, T. (1996). The yeast GALll protein binds to the transcription factor HE through GALll regions essential for its in vivo flmction. Proc Natl Acad Sci U S A 93, 9488-92. Sakurai, H., Ohishi, T., and Fukasawa, T. (1997). Promoter structm'e-dependent flmctioning of the general transcription factor HE in Saccharomyces cerevisiae. J Biol Chem 272, 15936-15942. Sarnkmashvili, I., and Luse, D. S. (1998). Structural changes in the RNA polymerase H transcription complex during transition fiom initiation to elongation. Mol Cell Biol 18, 5343-5354. Sancar, A. (1996). DNA excision repair. Annu Rev Biochem 65, 43-81. Sauer, P., Fondell, J. D., Ohkuma, Y., Roeder, R. G., and Jackle, H. (1995). Control of transcription by Kruppel through interactions with TFHB and TFHE beta. Nature 375, 162-4. Sauer, F., Hansen, S. K, and Tjian, R. (1995). DNA template and activator-coactivator requirements for transcriptional synergism by Drosophila bicoid. Science 270, 1825- 1828. Sawadogo, M., and Roeder, R. G. (1985a). Factors involved in specific transcription by human RNA polymerase H: analysis by a rapid and quantitative in vitro assay. Proc Natl Acad Sci U S A 82, 4394-8. Sawadogo, M., and Roeder, R. G. (1985b). Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region. Cell 43, 165-75. Sayre, M. H., Tschochner, H., and Kornberg, R. D. (1992). Reconstitution of transcription with five purified initiation factors and RNA polymerase H fiom Saccharomyces cerevisiae. J Biol Chem 26 7, 23376-82. Schaeffer, L., Moncollin, V., Roy, R., Staub, A., Mezzina, M., Sarasin, A., Weeda, G., Hoeijmakers, J. H., and Egly, J. M. (1994). The ERCC2/DNA repair protein is associated with the class H BTF2/I'FIIH transcription factor. EMBO J 13, 2388-92. 218 Schaefl‘er, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J. H., Chambon, P., and Egly, J. M. (1993). DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor. Science 260, 58-63. Schickor, P., Metzger, W., Werel, W., Lederer, H., and Heumann, H. (1990). Topography of intermediates in transcription initiation of E.coli. Embo J 9, 2215-2220. Schultz, P., Celia, H., Riva, M., Sentenac, A., and Oudet, P. (1993). Three-dimensional model of yeast RNA polymerase 1 determined by electron microscopy of two- dimensional crystals. Embo J 12, 2601-2607. Schwerk, C., Klotzbucher, M., Sachs, M., Ulber, V., and Klein Hitpass, L. (1995). Identification of a transactivation function in the progesterone receptor that interacts with the TAFH110 subunit of the TFHD complex. J Biol Chem 270, 21331-8. Scully, R., Anderson, S. P., Chao, D. M., Wei, W., Ye, L., Young, R. A., Livingston, D. M., and Parvin, J. D. (1997). BRCAl is a component of the RNA polymerase H holoenzyme. Proc Natl Acad Sci U S A 94, 5605-5610. Selby, C. P., Drapkin, R., Reinberg, D., and Sancar, A. (1997). RNA polymerase H stalled at a thymine dimer: footprint and efl‘ect on excision repair. Nucleic Acids Res 25, 787-93. Serizawa, H., Conaway, J. W., and Conaway, R. C. (1993). Phosphorylation of C- terminal domain of RNA polymerase H is not required in basal transcription. Nature 363, 371-374. Serizawa, H., Conaway, R. C., and Conaway, J. W. (1992). A carboxyl-terminal-domain kinase associated with RNA polymerase H transcription factor delta fiom rat liver. Proc Natl Acad Sci U S A 89, 7476-7480. Serrza' wa, H., Conaway, R. C., and Conaway, J. W. (1993). Multiftmctional RNA polymerase H initiation factor delta fiom rat liver. Relationship between carboxyl- terminal domain kinase, ATPase, and DNA helicase activities. J Biol Chem 268, 17300- 17308. Severinov, K, Mustaev, A., Kukarin, A., Muzzin, 0., Bass, 1., Darst, S. A., and Goldfarb, A. (1996). Structural modules of the large subunits of RNA polymerase. Introducing archaebacteria] and chloroplast split sites in the beta and beta' subunits of Escherichia coli RNA polymerase. J Biol Chem 271, 27969-74. Shapiro, D. J., Sharp, P. A., Wahli, W. W., and Keller, M. J. (1988). A high-efficiency HeLa cell nuclear transcription extract. DNA 7, 47-55. Sheri, W. C., and Green, M. R. (1997). Yeast TAF(H)145 functions as a core promoter selectivity factor, not a general coactivator. Cell 90, 615-624. 219 Shi, X., Chang, M., Wolf, A. J., Chang, C. H., Frazer Abel, A. A., Wade, P. A., Burton, Z. P., and Jaehning, J. A. (1997). Cdc73p and Paflp are found in a novel RNA polymerase H-containing complex distinct fiom the Srbp-containing holoenzyme. Mol Cell Biol 17, 1 160-9. Shi, X., Finkelstein, A., W01f, A. J ., Wade, P. A., Burton, Z. P., and Jaehning, J. A. (1996). Pafl p, an RNA polymerase H-associated factor in Saccharomyces cerevisiae, may have both positive and negative roles in transcription. Mol Cell Biol 16, 669-76. Shilatifard, A., Duan, D. R., Haque, D., Florence, C., Schubach, W. H., Conaway, J. W., and Conaway, R. C. (1997a). ELL2, a new member of an ELL family of RNA polymerase H elongation factors. Proc Natl Acad Sci U S A 94, 3639-3643. Shilatifard, A., Haque, D., Conaway, R. C., and Conaway, J. W. (1997b). Structure and frmction of RNA polymerase H elongation factor ELL. Identification of two overlapping ELL ftmctional domains that govern its interacn'on with polymerase and the ternary elongation complex. J Biol Chem 272, 22355-22363. Shilatifard, A., Lane, W. S., Jackson, K W., Conaway, R. C., and Conaway, J. W. (1996). An RNA polymerase H elongation factor encoded by the human ELL gene. Science 271, 1873-1876. Shykind, B. M., Kim, J ., and Sharp, P. A. (1995). Activation of the TFHD-TFIIA complex with I-IMG-2. Genes Dev 9, 1354-65. Singer, V. L., Wobbe, C. R., and Struhl, K (1990). A wide variety of DNA sequences can flmctionally replace a yeast TATA element for transcriptional activation. Genes Dev 4, 636-45. Smale, S. T. (1997). Transcription initiation fiom TATA-less promoters within eukaryotic protein-coding genes. Biochim Biophys Acta 1351, 73-88. Smale, S. T., Schmidt, M. C., Berk, A. J ., and Baltimore, D. (1990). Transcriptional activation by Spl as directed through TATA or initiator: specific requirement for mammalian transcription factor HD. Proc Natl Acad Sci U S A 87, 4509-13. Sopta, M., Burton, Z. R, and Greenblatt, J. (1989). Structure and associated DNA- helicase activity of a general transcription initiation factor that binds to RNA polymerase H. Name 341, 410-4. Sopta, M., Carthew, R. W., and Greenblatt, J. (1985). Isolation of three proteins that bind to mammalian RNA polymerase H. J Biol Chem 260, 10353-60. Steinmetz, E. J. (1997). Pro-mRNA processing and the CTD of RNA polymerase H: the tail that wags the dog? Cell 89, 491-494. 220 Sumimoto, H., Ohkuma, Y., Sinn, E., Kato, H., Shimasaki, S., Horikoshi, M., and Roeder, R. G. (1991). Conserved sequence motifs in the small subunit of human general transcription factor TFHE. Nature 354, 401-4. Stm, X., Ma, D., Sheldon, M., Yeung, K, and Reinberg, D. (1994). Reconstitution of human TFHA activity fiorn recombinant polypeptides: a role in TFHD-mediated transcription. Genes Dev 8, 2336-48. Sun, Z. W., and Hampsey, M. (1995). Identification of the gene (SSU7 l/TF G1) encoding the largest subunit of transcription factor TFIIF as a suppressor of a TFHB mutation in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 92, 3127-31. Sun, Z. W., Tessmer, A., and Hampsey, M. (1996). Functional interaction between TFHB and the pr9 (Ssu73) subunit of RNA polymerase H in Saccharomyces cerevisiae. Nucleic Acids Res 24, 2560-6. Szentirmay, M. N., and Sawadogo, M. (1994). Sarkosyl block of transcription reinitiation by RNA polymerase H as visualized by the colliding polymerases reinitiation assay. Nucleic Acids Res 22, 5341-6. Taggart, A. K, Fisher, T. S., and Pugh, B. F. (1992). The TATA-binding protein and associated factors are components of pol HI transcription factor TFHIB. Cell 71 , 1015-28. Takagi, Y., Conaway, R. C., and Conaway, J. W. (1996). Characterization of elongin C functional domains required for interaction with elongin B and activation of elongin A. J Biol Chem 271, 25562-25568. Takagi, Y., Pause, A., Conaway, R. C., and Conaway, J. W. (1997). Identification of elongin C sequences required for interaction with the von Hippel-Lindau tumor , suppressor protein. J Biol Chem 272, 27444-27449. Tan, 8., Aso, T., Conaway, R. C., and Conaway, J. W. (1994). Roles for both the RAP30 and RAP74 subunits of transcription factor IIF in transcription initiation and elongation by RNA polymerase H. J Biol Chem 269, 25684-91. Tan, S., Conaway, R. C., and Conaway, J. W. (1995). Dissection of transcription factor TFHF functional domains required for initiation and elongation. Proc Natl Acad Sci U S A 92, 6042-6046. Tan, 8., Garrett, K P., Conaway, R. C., and Conaway, J. W. (1994). Cryptic DNA- binding domain in the C terminus of RNA polymerase H general transcription factor RAP30. Proc Natl Acad Sci U S A 91, 9808-12. Tan, 8., Hunziker, Y., Sargent, D. F., and Richmond, T. J. (1996). Crystal structm'e of a yeast TFHA/TBP/DNA complex. Name 381, 127-151. 221 Tanese, N., Pugh, B. F., and Tjian, R. (1991). Coactivators for a proline-rich activator purified fiom the multisubunit human TFIID complex. Genes Dev 5, 2212-24. Taunton, J ., Hassig, C. A., and Schreiber, S. L. (1996). A mammalian histone deacetylase related to the yeast transcriptional regulator de3p. Science 272, 408-411. 1 Thompson, C. M., Koleske, A. J ., Chao, D. M., and Young, R. A. (1993). A multisubunit complex associated with the RNA polymerase H CTD and TATA-binding protein in yeast. Cell 73, 1361-75. Thompson, C. M., and Young, R. A. (1995). General requirement for RNA polymerase II holoenzymes in vivo.‘ Proc Natl Acad Sci U S A 92, 4587-90. Tong, X., Drapkin, R., Yalamanchili, R., Mosialos, G., and Kieff, E. (1995). The Epstein- Barr virus nuclear protein 2 acidic domain forms a complex with a novel cellular coactivator that can interact with TFHE. Mol Cell Biol 15, 4735-44. Travers, A. A. (1990). Why bend DNA? Cell 60, 177-180. Triezenberg, S. J. (1995). Structure and function of transcriptional activation domains. Cm'r Opin Genet Dev 5, 190-196. Truant, R., Xiao, H., Ingles, C. J., and Greenblatt, J. (1993). Direct interaction between the transcriptional activation domain of human p53 and the TATA box-binding protein. J Biol Ch- 268, 2284-7. Tsukiyama, T., and Wu, C. (1995). Purification and properties of an ATP-dependent nucleosome remodeling factor. Cell 83, 1011- 1020. Tyree, C. M., George, C. P., Lira-DeVito, L. M., Wampler, S. L., Dahmus, M. E., Zawel, L., and Kadonaga, J. T. (1993). Identification of a minimal set of proteins that is suficient for accurate initiation of transcription by RNA polymerase H. Genes Dev 7, 1254-1265. Ucker, D. S, and Yamamoto, K R. (1984). Early events in the stimulation of mammary tumor virus RNA synthesis by glucocorticoids. Novel assays of transcription rates. J Biol Chem 259, 7416-7420. Usheva, A., Maldonado, E., Goldring, A., Lu, H., Houbavi, C., Reinberg, D., and Aloni, Y. (1992). Specific interaction between the nonphosphorylated form of RNA polymerase H and the TATA-binding protein. Cell 69, 871-81. Usheva, A., and Shenk, T. (1994). TATA-binding protein-independent initiation: YYl , TFHB, and RNA polymerase H direct basal transcription on supercoiled template DNA. Cell 76, 1115-21. 222 Van Dyke, M. W., Roeder, R. G., and Sawadogo, M. (1988). Physical analysis of transcription preinitiation complex assembly on a class H gene promoter. Science 241 , 1335-8. van Vum'en, A. J ., Vermeulen, W., Ma, L., Weeda, G., Appeldoorn, E., Jaspers, N. G., van der Eb, A. J ., Bootsma, D., Hoeijmakers, J. H., Humbert, S., and et al. (1994). Correction of xeroderma pigmentosum repair defect by basal transcription factor BTF 2 (TFHH). EMBO J 13, 1645-53. Varga-Weisz, P. D., Wilm, M., Bonte, E., Dumas, K, Mann, M., and Becker, P. B. (1997). Chromatin-remodelling factor CI-IRAC contains the ATPases ISWI and topoisomerase H. Nature 388, 598-602. Verrijzer, C. P., Chen, J. L., Yokomori, K, and Tjian, R. (1995). Binding of TAFs to core elements directs promoter selectivity by RNA polymerase H. Cell 81 , 1115-25. Verrijzer, C. P., and Tjian, R. (1996). TAFs mediate transcriptional activation and promoter selectivity. Trends Biochem Sci 21 , 338-342. Verrijzer, C. P., Yokomori, K, Chen, J. L., and Tjian, R. (1994). Drosophila TAFH150: similarity to yeast gene TSM-l and specific binding to core promoter DNA. Science 264, 933-41 . Vidal, M., and Gaber, R. F. (1991). RPD3 encodes a second factor required to achieve maximum positive and negative transcriptional states in Saccharomyces cerevisiae. Mol Cell Biol 11, 6317-6327. von Hippel, P. H. (1998). An integrated model of the transcription complex in elongation, termination, and editing. Science 281, 660-665. von Hippel, P. H., and Yager, T. D. (1992). The elongation-termination decision in transcription. Science 255, 809-812. Wade, P. A., Werel, W., Fentzke, R. C., Thompson, N. E., Leykam, J. F., Burgess, R. R., Jaehning, J. A., and Burton, Z. F. (1996). A novel collection of accessory factors associated with yeast RNA polymerase H. Protein Expr Pmif 8, 85-90. Walker, S. 8., Reese, J. C., Apone, L. M., and Green, M. R. (1996). Transcription activation in cells lacking TAFHS. Name 383, 185-188. Walker, S. S., Shen, W. C., Reese, J. C., Apone, L. M., and Green, M. R. (1997). Yeast TAF(H)145 required for transcription of G1/S cyclin genes and regulated by the cellular growth state. Cell 90, 607-614. 223 Wampler, S. L., and Kadonaga, J. T. (1992). Functional analysis of Drosophila transcription factor HB. Genes Dev 6, 1542-52. Wang, B. Q., and Burton, Z. P. (1995). Functional domains of hmnan RAP74 including a masked polymerase binding domain. J Biol Chem 270, 2703 5-44. Wang, B. Q., Kostrub, C. F., Finkelstein, A., and Burton, Z. F. (1993). Production of human RAP30 and RAP74 in bacterial cells. Protein Expr Pmif 4, 207-14. Wang, B. Q., Lei, L., and Burton, Z. F. (1994). Importance of codon preference for production of human RAP74 and reconstitution of the RAP30/74 complex. Protein Expr Pmif 5, 476-85. Wang, E. H., Zou, S., and Tjian, R. (1997). TAFH250-dependent transcription of cyclin A is directed by ATP activator proteins. Genes Dev 11, 2658-2669. Wang, W., Carey, M., and Gralla, J. D. (1992). Polymerase H promoter activation: closed complex formation and ATP- driven start site opening. Science 255, 450-453. Wang, W., Gralla, J. D., and Carey, M. (1992). The acidic activator GAL4-AH can stimulate polymerase H transcription by promoting assembly of a closed complex requiring TFHD and TFHA. Genes Dev 6, 1716-27. Wei, P., Garber, M. E., Fang, S. M., Fischer, W. H., and Jones, K A. (1998). A novel CDK9-associated C-type cyclin interacts directly with I-HV-l Tat and mediates its high- affinity, loop-specific binding to TAR RNA. Cell 92, 451-462. Weil, P. A., Luse, D. S., Segall, J., and Roeda’, R. G. (1979). Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on pmified RNA polymerase H and DNA. Cell 18, 469-484. Weis, L., and Reinberg, D. (1992). Transcription by RNA polymerase H: initiator- directed formation of transcription-competent complexes. FASEB J 6, 3300-9. White, J., Brou, C., Wu, J., Lutz, Y., Moncollin, V., and Chambon, P. (1992). The acidic transcriptional activator GAL-VP16 acts on preformed template-committed complexes. EMBO J 11, 2229-40. White, R. J ., and Jackson, S. P. (1992). The TATA-binding protein: a central role in transcription by RNA polymerases I, H and HI. Trends Genet 8, 284-8. Wieczorek, E., Brand, M., Jacq, X., and Tora, L. (1998). Function of TAF(H)-containing complex without TBP in transcription by RNA polymerase H. Nature 393, 187-191. 224 Wilson, C. J., Chao, D. M., Imbalzano, A. N., Schnitzler, G. R., Kingston, R. E., and Yormg, R. A. (1996). RNA polymerase H holoenzyme contains SWI/SNF regulators involved in chromatin remodeling. Cell 84, 23 5-44. Wobbe, C. R., and Struhl, K (1990). Yeast and human TATA-binding proteins have nearly identical DNA sequence requirements for transcription in vitro. Mol Cell Biol 10, 3859-67. Workman, J. L., Taylor, I. C., and Kingston, R. E. (1991). Activation domains of stably bound GAL4 derivatives alleviate repression of promoters by nucleosomes. Cell 64, 533- 44. Wu, S. Y., Kershnar, E., and Chiang, C. M. (1998). TAFH-independent activation mediated by human TBP in the presence of the positive cofactor PC4. Embo J 1 7, 447 8- 4490. - Wu, Y., Reece, R. J., and Ptashne, M. (1996). Quantitation of putative activator-target afiinities predicts transcriptional activating potentials. EMBO J 15, 3951-63. Xiao, H., Friesen, J. D., and Lis, J. T. (1995). Recruiting TATA-binding protein to a promoter: transcriptional activation without an upstream activator. Mol Cell Biol 15, 5757-61 . Xiao, H., Lis, J. T., and Jeang, K T. (1997). Promoter activity of Tat at steps subsequent to TATA-binding protein recruitment. Mol Cell Biol 1 7, 6898-6905. Xiao, H., Pearson, A., Coulombe, B., Truant, R., Zhang, S., Regier, J. L., Triezenberg, S. J., Reinberg, D., Flores, O., Ingles, C. J ., and et al. (1994). Binding of basal transcription factor TFHH to the acidic activation domains of VP16 and p53. Mol Cell Biol 14, 7013- 24. Xie, X., Kokubo, T., Cohen, s. L., Mirza, U. A., Hoffinann, A., Chait, B. T., Roeder, R. G., Nakatani, Y., and Burley, S. K (1996). Structural similarity between TAFs and the heterotetrameric core of the histone octamer. Nature 380, 316-22. Xie, Z., and Price, D. (1997). Drosophila factor 2, an RNA polymerase H transcript release factor, has DNA-dependent ATPase activity. J Biol Chem 272, 31902-31907. Xie, Z., and Price, D. H. (1996). Pmification of an RNA polymerase H transcript release factor from Drosophila. J Biol Chem 271, 11043-11046. Xing, L., Gopal, V. K, and Quinn, P. G. (1995). cAMP response element-binding protein (CREB) interacts with transcription factors HB and HD. J Biol Chem 270, 17488-17493. 225 Yamashita, S., Hisatake, K, Kokubo, T., Doi, K, Roeder, R. G., Horikoshi, M., and Nakatani, Y. (1993). Transcription factor TFHB sites important for interaction with promoter-bound TFHD. Science 261 , 463-6. Yamashita, S., Wada, K, Horikoshi, M., Gong, D. W., Kokubo, T., Hisatake, K, Yokotani, N., Malik, S., Roeder, R. G., and Nakatani, Y. (1992). Isolation and characterization of a cDNA encoding Drosophila transcription factor TFHB. Proc Natl Acad Sci U S A 89, 2839-43. Yang, X. J ., Ogryzko, V. V., Nishikawa, J., Howard, B. H., and Nakatani, Y. (1996). A p300/CBP-associated factor that competes with the adenoviral oncoprotein ElA. Nature 382, 319-324. Yankulov, K, Blau, J ., Purton, T., Roberts, 8., and Bentley, D. L. (1994). Transcriptional elongation by RNA polymerase H is stimulated by transactivators. Cell 7 7, 749-759. Yankulov, K, Yamashita, K, Roy, R., Egly, J. M., and Bentley, D. L. (1995). The transcriptional elongation inhibitor 5,6-dichloro-1-beta-D-ribofirranosylbenzimidazole inhibits transcription factor [Hi-associated protein kinase. J Biol Chem 270, 23922-5 . Yokomori, K, Admon, A., Goodrich, J. A., Chen, J. L., and Tjian, R. (1993). Drosophila TFHA-L is processed into two subunits that are associated with the TBP/TAF complex. Genes Dev 7, 2235-45. Yokomori, K, Zeidler, M. P., Chen, J. L., Verrijzer, C. P., Mlodzik, M., and Tjian, R. (1994). Drosophila TFHA directs cooperative DNA binding with TBP and mediates transcriptional activation. Genes Dev 8, 2313-23. Yonaha, M., Aso, T., Kobayashi, Y., Vasavada, H., Yasukochi, Y., Weissman, S. M., and Kitajima, S. (1993). Domain structme of a human general transcription initiation factor, TFHF. Nucleic Acids Res 21 , 273-9. Yoon, H., Sitikov, A. S., Jeon, C., and Agarwal, K (1998). Preferential interaction of the mRNA proofi'eading factor TFIIS zinc ribbon with rU.dA base pairs correlates with its function. Biochemistry 37, 12104-12112. Young, R. A. (1991). RNA polymerase H. Annu Rev Biochem 60, 689-715. Yuan, C. X., Ito, M., Fondell, J. D., Fu, Z. Y., and Roeder, R. G. (1998). The TRAP220 component of a thyroid hormone receptor- associated protein (TRAP) coactivator ‘ complex interacts directly with nuclear receptors in a ligand-dependent fashion. Proc Natl Acad Sci U S A 95, 7939-7944. ’ Yue, Z., Maldonado, E., Pillutla, R., Cho, H., Reinberg, D., and Shatkin, A. J. (1997). Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking 226 mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase H. Proc Natl Acad Sci U S A 94, 12898-12903. Zandomeni, R., Bunick, D., Ackerman, S., Mittleman, B., and Weinmann, R. (1983). Mechanism of action of DRB. HI. Effect on specific in vitro initiation of transcription. J Mol Biol 167, 561-574. Zandomeni, R., and Weinmann, R. (1984). Inhibitory effect of 5,6-dichloro-l-beta-D- ribofuranosylbenzimidazole on a protein kinase. J Biol Chem 259, 14804-14811. Zawel, L., Kumar, K P., and Reinberg, D. (1995). Recycling of the general transcription factors during RNA polymerase H transcription. Genes Dev 9, 1479-90. Zawel, L., and Reinberg, D. (1995). Common themes in assembly and flmction of eukaryotic transcription complexes. Annu Rev Biochem 64, 533-61. Zawel, L., and Reinberg, D. (1993). Initiation of transcription by RNA polymerase H: a multi-step process. Prog Nucleic Acid Res Mol Biol 44, 67-108. Zaychikov, E., Martin, E., Denissova, L., Kozlov, M., Markovtsov, V., Kashlev, M., Heumann, H., Nikiforov, V., Goldfarb, A., and Mustaev, A. (1996). Mapping of catalytic residues in the RNA polymerase active center. Science 273, 107-9. Zhang, Y., Iratni, R, Erdjument-Bromage, H., Tempst, P., and Reinberg, D. (1997). Histone deacetylases and SAP 1 8, a novel polypeptide, are components of a human Sin3 complex. Cell 89, 357-364. ~ Zhou, Q., Boyer, T. G., and Berk, A. J. (1993). Factors (TAFs) required for activated transcription interact with TATA box-binding protein conserved core domain. Genes Dev 7, 180-7 . Zhou, Q., Lieberman, P. M., Boyer, T. G., and Berk, A. J. (1992). Holo-TFHD supports transcriptional stimulation by diverse activators and fiorn a TATA-less promoter. Genes Dev 6, 1964-74. Zhu, A., and Kuziora, M. A. (1996). Homeodomain interaction with the beta subunit of the general transcription factor TFHE. J Biol Chem 271, 20993-6. Zhu, H., Joliot, V., and Prywes, R. (1994). Role of transcription factor TFHF in serum response factor-activated transcription. J Biol Chem 269, 3489-97. Zhu, W., Zeng, Q., Colangelo, C. M., Lewis, M., Summers, M. F., and Scott, R. A. (1996). The N-terminal domain of TFIIB fiorn Pyrococcus fmiosus forms a zinc ribbon. Nat Struct Biol 3, 122-124. 227 Zhu, Y., Pe'ery, T., Peng, J., Ramanathan, Y., Marshall, N., Marshall, T., Amendt, B., Mathews, M. B., and Price, D. H. (1997). Transcription elongation factor P-TEFb is required for I-HV-l tat transactivation in vitro. Genes Dev 11, 2622-2632. Zwilling, S., Annweiler, A., and Wirth, T. (1994). The POU domains of the Octl and Oct2 transcription factors mediate specific interaction with TBP. Nucleic Acids Res 22, 1655—62. 228