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This is to certify that the

dissertation entitled

Functions of Transcription Factor IIF
in Initiation and Elongation by RNA Polymerase II

presented by

Chun—hsiang Chang

has been accepted towards fulfillment
of the requirements for

Ph.D. Biochemistry

 

 

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FUNCTIONS OF TRANSCRIPTION FACTOR IIF
IN INITIATION AND ELONGATION BY RNA POLYMERASE II

BY

Chun-hsiang Chang

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1995

ABSTRACT

FUNCTIONS OF TRANSCRIPTION FACTOR IIF
IN INITIATION AND ELONGATION BY RNA POLYMERASE II

BY

Chun-hsiang Chang

Understanding RNA polymerase II-dependent transcription requires
characterization of basal and regulatory factors and their participation in transitions
between stages of the transcription cycle. This study focuses on functions of general
transcription factor TFIIF and the transition from initiation to productive elongation. A
role for the large TFIIF subunit (named RAP74 for RNA polymerase II-associated
protein; 74 kilodaltons) in this transition is demonstrated. An assay for detection of the
first stable transcription complexes initiated from the Adenovirus major late promoter has
been developed and applied to understand functions of RAP74 in maintenance of short
transcript stability.

Both RAP30 and RAP74 subunits of TFIIF are required for accurate transcription
in vitro. Here we demonstrate that in some in vitro systems the RAP30 subunit is
required for accurate initiation, but the RAP74 subunit is not. RAP74, however, is
required for transition to a productive elongation complex. Thus, the two subunits of
TFIIF have partially separable functions in initiation and early elongation of
transcription.

To better understand the transition between initiation and elongation by RNA

polymerase 11, an assay system was designed in which the Adenovirus major late

promoter was immobilized on agarose beads. The first stable transcripts initiated on
these immobilized templates were isolated and analyzed. A kinetic lag of 15 to 20 s was
observed before stable transcripts appeared when nucleoside triphosphates were added to
pre-formed transcription complexes. Depending on the nucleoside triphosphate that is
limiting in concentration, RNA polymerase II pauses at various positions between
nucleotide +11 to +20 of the RNA chain. Transcripts shorter than 11 nucleotides in
length are not stably bound to the beads, and must be released as abortive transcripts.
Short, paused RNAs are stable to washing with buffer and can be quantitatively chased to
the runoff position on the template.

The stability of short RNA complexes is compromised if transcription is initiated
in the presence of certain RAP74 mutants. Human RAP74 is a 517 amino acid protein.
The C-tcrminal region of RAP74, including amino acids 409-517, contributes to short
transcript stability. A 1-172 mutant, previously shown in another assay to be inactive for
accurate transcription, is shown here to support accurate intiation. Transcripts initiated in
the presence of this mutant are very unstable, compared to a 1-205 mutant. The role of
RAP74 sequences in stabilizing short ternary complexes may partially explain the role of
RAP74 in promoter escape.

In the final chapter of this thesis, preliminary experiments are shown in which a
yeast protein Cdc73p is demonstrated to bind directly to RNA polymerase H. Cdc73p is
of interest because from sequence analysis this protein appears to be a subunit of an
alternate form of TFIIF. Cdc7 3p has similar structures to human RAP30, bacterial sigma
factors, and bacterial delta factor. These similarities were used to predict the region of
Cdc73p that might bind to RNA polymerase II. Deletion of 15 amino acids from this

region severely inhibited polymerase binding.

Dedicated to my friends, my teachers, and my family.

iv

ACKNOWLEDGMENTS

I would like to express my appreciation to my mentor, Dr. Zachary Burton, for his
guidance ,support and encouragement through the years. I wish to thank members of my
guidance committee, Drs. William Helferich, Rawle Hollingsworth, Arnold Revzin and

William Smith for their advise and encouragement.

I would like to acknowledge the support of my colleagues, Dr. Ann Finkelstein,
Dr. Wladyslaw Werel, David Chavez, Shimin Fang, Richard Fentzke, Jenny Jensen,
Corwin Kostrub, Lei Lei, Augie Pioszak, Stephan Reymez, Bo Qing Wang, and Yong
Wang. Especially, I want to thank Cory and Bo Qing for the recombinant RAP30 and
RAP74 proteins that they have provided in my study.

Finally , I am grateful to my parents for everything they have given to me and to

my wife for her understanding and unconditional support.

TABLE OF CONTENTS

PAGE

LIST OF FIGURES .................................................................................................. x

LIST OF ABBREVIATIONS .................................................................................. xii
CHAPTER I: INTRODUCTION ................................................................. 1
Literature Review ......................................................................................... 2
Transcription in Prokaryotes ............................................................ 2
Transcription by RNA Polymerase II ............................................... 5
General Transcription Factors .............................................. 6
TFIID ........................................................................ 6
TFIIA ........................................................................ 7
TFIIB ........................................................................ 8
TFIIF ........................................................................ 9

TFIIE ........................................................................ 1 1

TFIIH ........................................................................ 1 1

RNA polymerase II and RAPs ......................................................... 12

RNA polymerase II .............................................................. 12

RAPs ..................................................................................... 14

Mechanism of RNA Polymerase H Transcription ............................ 16

1) Template activation .......................................................... 16

2) Promoter recognition ........................................................ 17

(I) Multiple-step assembly process ........................... 18

(II) RNAP II holoenzyme complexes ....................... 18

(III) Minimal transcription systems .......................... 22

3) Open complex formation ................................................. 22

4) Abortive initiation and promoter escape .......................... 23

5) Elongation ........................................................................ 25

6) Termination ...................................................................... 28

7)Recycling of RNAP II ....................................................... 29

Response of the general mechanism to activators ............................ 29

Overview ...................................................................................................... 30

References .................................................................................................... 32

CHAPTER II RAP30/74 (TFIIF) IS REQUIRED FOR

PROMOTER ESCAPE BY RNA POLYMERASE 11.... 44
Abstract ........................................................................................................ 45
Introduction .................................................................................................. 46
Material and Methods ................................................................................... 49
DNA templates for transcription assays ........................................... 49
Extract systems for in vitro transcription ......................................... 49
Recombinant RAP30 and RAP74 .................................................... 49
In vitro transcription assays .............................................................. 50
Results .......................................................................................................... 51

RAP30 and RAP74 are required for accurate
transcription from the adenovirus major late promoter .................... 51

RAP30 and RAP74 are required for accurate
transcription in the presence of sarkosyl .......................................... 54
Requirement for RAP30/74 function before sarkosyl addition ........ 57
RAP74 is dispensable for initiation but required for elongation ...... 6O

Confirmation of the sarkosyl results using a pulse-chase protocol.. 64

Discussion .................................................................................................... 7O
Acknowledgments ........................................................................................ 77
References .................................................................................................... 78

CHAPTER III: STABILITY OF TERNARY COMPLEXES:

STALLING OF RNA POLYMERASE II

NEAR THE TRANSCRIPTIONAL START SITE ......... 81

Abstract ........................................................................................................ 82
Introduction .................................................................................................. 83
Material and Methods ................................................................................... 87
Construction of the Immobilized templates ..................................... 87
Transcription assays ......................................................................... 87
Results .......................................................................................................... 88

The stalling of RNA polymerase II can be observed with

vii

different radioactive labeling and the precise pause

is dependent on which NTP is limiting ............................................ 88
Stalling of RNA polymerase II is observed with

less limitation of radioactive NTP .................................................... 93
+116 synthesis is dependent upon ATP hydrolysis ......................... 96
Catalytic sites of RNA polymerase II location and precise

sizes of short RN As are determined by conditional chase ............... 99
Runoff RNA can be synthesized from

the paused ternary complexes ........................................................... 102

Preinitiation complexes can be isolated by immobilized template. 102
A 15 to 20 second kinetic lag is observed in formation

of stable ternary complex from the AdML promoter ....................... 102
RAP74 contributes to the stability of ternary complexes ................. 107
Discussion .................................................................................................... 1 14
Acknowledgments ........................................................................................ 120
References .................................................................................................... 121

CHAPTER IV: RNA POLYMERASE II BINDING

OF YEAST CDC73P .......................................................... 124

Abstract ........................................................................................................ 125

Introduction .................................................................................................. 126

Material and Methods ................................................................................... 128

Expression vector and strains ........................................................... 128
Production and purification of Cdc73p,

cdc73p A129-149 and cdc73p 1-143 ................................................ 128

Production of anti-Cdc73p antiserum ............................................... 128

Gel filtration chromatography binding assay ................................... 129

Anclp binding assay ........................................................................ 129

Results .......................................................................................................... 130

Cdc7 3p is associated with yeast RNA polymerase II ....................... 130

Cdc73p may share sequence similarity to
human RAP30 and bacterial 0 factors
within their RNA polymerase binding regions ................................. 130

viii

Production and purification of Cdc73p and

Cdc73p mutant proteins .................................................................... 135

The predicted interaction region of Cdc73p

is important for RNA polymerase 1] binding ................................... 135

Cdc73p may interact with the small subunit of yeast TFIIF ............ 142
Discussion .................................................................................................... 145
Acknowledgment. ......................................................................................... 149
References .................................................................................................... 150

Figure 1.

Figure 1.

Figure 2.

Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.

Figure 1.
Figure 2.

Figure 3.

Figure 4.
Figure 5.
Figure 6.
Figure 7.

Figure 8.
Figure 9.

LIST OF FIGURES

PAGE
Chapter I
Preinitiation complex formation pathways ....................................... 19
Chapter II
Both RAP30 and RAP74 are required for
accurate transcription from the AdMLP ........................................... 52
Both RAP30 and RAP74 are required for
accurate transcription in the presence of sarkosyl ............................ 55
Kinetics of RAP30 and RAP74 function in transcription ................ 58
RAP74 is an elongation factor .......................................................... 62
Pulse-chase protocol: RAP30 is an initiation factor ......................... 65
Pulse-chase protocol: RAP74 is an elongation factor ...................... 68
A model for RAP30/74 function in initiation
and early elongation of transcription ................................................ 71
Chapter III
Immobilized template ....................................................................... 89
Synthesis of short transcripts by different labeling
of the short transcripts from AdML promoter .................................. 91
Paused transcripts are also observed in
less limitation of radioactive NTP .................................................... 94
Short transcripts synthesis are dependent on ATP hydrolysis ......... 97
Conditional chase from +1 16 of the AdML transcript .................... 100
Short transcripts are the precursors of runoff RNA ......................... 103
Washes of the preinitiation complexes do not
alter the stalling of RNA polymerase II ........................................... 105
A 15 to 20 seconds lag in stable ternary complexes synthesis ......... 108
RAP74 is important for the stability
of initiated transcription complexes ................................................. 111

Figure 10.

Figure 1.
Figure 2.

Figure 3.
Figure 4.

Figure 5.

Figure 6.
Figure 7.

A model describing the role of RAP74 in accurate

initiation and promoter escape by RNA polymerase H .................... 117
Chapter IV
Cdc73p is associated with yeast RNA polymerase H ....................... 131

Cdc73p may share sequence similarity to
human RAP30 and bacterial 0 factors

within their RNA polymerase binding regions ................................. 133
Cdc73p mutant proteins .................................................................... 136
Production and purification of Cdc73p mutant proteins .................. 138

The predicted interaction region of Cdc73p
is important for RNA polymerase II binding ................................... 140

Cdc73p interacts with Anclp/ng3p ................................................ 143

Proposed similarities between yeast Cdc73p,
human RAP30, and bacterial sigma and delta factors ...................... 146

a-A
A280

A600

AdMLP

ATP

BSA

Da
DNA
D'I'I‘
EDTA

EGTA
FPLC
GTP
HCA
HEPES

kb
kDa
LB

mRNA

LIST OF ABBREVIATIONS
a-amanitin
absorbance at 280 nm
absorbance at 600 nm
amino acid(s)
Adenovirus major late promoter
AMPPNP
adenosine triphosphate
base pairs
bovine serum albumin
cytosine triphosphate
Dalton
deoxyribonucleic acid
dithiothreitol
ethylenediamine tetraacetic acid
ethyleneglycol-bis-(B—aminoethyl ether) N,N,N',N'-tetraacetic acid
fast protein liquid chromatography
guanine triphosphate
hydrophobic cluster analysis
N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid)
isopropyl-BoD-thiolgalactopyranoside
kilobase pairs
kilodaltons
Luria-Bertani
3'-O-methyl GTP

messenger RNA

xii

nt

OD
PAGE

RNAP II

rRNA
SDS
TAF
TBP
TFH
tRNA

Single letter abbreviations for the amino acids: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe;
G, Gly; H, His; 1, lle; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T,

HeLa nuclear extract

nucleotides

nucleoside triphosphate

optical density

polyacrylamide gel electrophoresis
polymerase chain reaction

RNA polymerase H

RNA polymerase H associating proteins
ribonucleic acid

ribosomal RNA

sodium dodecyl sulfate

TBP associated factor

TATA box-bindin g protein
transcription factor of RNA polymerase H

transfer RNA

Thr, V, Val; W, Trp; and Y, Tyr.

xiii

CHAPTER I

INTRODUCTION

2
LITERATURE REVIEW

Regulation of transcription is a critical stage of differentiation and development in
biological systems. A single form of RNA polymerase performs transcription of
ribosomal RNA (rRNA), messenger RNA (mRNA) and transfer RNA (tRNA) in
prokaryotes, whereas RNA polymerase I (RNAP 1), RNA polymerase H (RNAP H) and
RNA polymerase HI (RN AP IH), respectively, perform these functions in eukaryotes. An
important mechanism for controlling protein levels in cells is to regulate synthesis of
mRNA. Therefore, regulation of RNAP II is important to control growth and
differentiation of eukaryotic cells. Understanding the mechanism of transcription by
RNAP II is necessary to understand how this enzyme cooperates with regulators,
coactivators and basal factors to synthesize mRNA.

Transcription in prokaryotes

RNA synthesis mechanisms and regulation in prokaryotes provide important
models for understanding these processes in eukaryotes. The prokaryotic apparatus
includes the RNA polymerase core enzyme and a sigma factor. This holoenzyme has the
capacity to initiate RNA synthesis accurately from a promoter DNA sequence. Different
holoenzymes are distinguished by their associated sigma factors and specifically
transcribe genes under control of a specific set of promoters. For instance, most
promoters in E. coli are recognized by 670. This initiation factor has an RNA
polymerase binding domain and sequence-specific DNA-binding domains recognizing
the -10 (TATAAT) and -35 ('ITGACA) regions of promoters. When the sequence of the
promoter does not match the consensus, DNA-binding activator proteins are needed in
addition to 0’70 for efficient initiation. 0'70 first binds to RNA polymerase before
holoenzyme associates with the promoter. Binding of 070 releases RNA polymerase

from non-specific sites on DNA, and also alters the conformation of 070 to expose

3
hexamer DNA-binding domains (Gross et al., 1992; Drombowski et al., 1992). Other

sigma factors recognize distinct classes of promoters.

Initiation of transcription can be roughly divided into specific binding,
isomerization, abortive initiation and promoter escape. In the presence of its sigma
factor, RNA polymerase binds tightly to promoter DNA to form the closed complex. The
simultaneous interaction of RNAP with the —35 and -10 hexamers induces distortion of
the DNA (Mecsas et al., 1991); RNAP binding alters the torsion between the -35 and -10
sequences altering the twist of the spacer sequence (Ayer et al., 1989). The closed
complex isomerizes into an open complex by separating the template DNA strands.
Strand separation may initiate from the release of the torsional stress accumulated within
the spacer region. Abortive initiation occurs at many promoters. RNAP synthesizes
numerous short transcripts before entering a productive elongation mode (reviewed by
von Hippel et al., 1984; McClure, 1985). This process involves repeated re-initiation by
RNAP without escape from the promoter.

RN AP has two RNA binding sites within its active site (Reynolds et al., 1992). A
"loose" RNA binding site at the catalytic center where phosphodiester bonds are formed,
and a "tight" RNA binding site about 1012 nucleotides upstream of the catalytic center.
Abortive products are formed when RNA is too short to fill the loose binding site (Surratt
et al., 1991; Borukhov et al., 1993; Mustaev et al., 1993; Mustaev et al., 1994). Abortive
initation occurs when the high affinity of holoenzyme for the promoter out competes
productive elongation. A change in RNA polymerase conformation causes release of O70
and allows promoter escape and productive elongation (von Hippel et al., 1992; Gill et
al., 1991).

Elongation and termination of transcription are competitive processes. The
probability of continuing or terminating a chain is dependent on the conformation of
RNAP, interactions with RNA bound in the "tight" and "loose" sites, and interaction with

the template. Termination is likely to require dissociation of RNA from the tight binding

4
site. The decision to elongate or terminate is influenced by several mechanisms: 1)

modification of RN AP into a terminating or anti-terminating form by binding accessory
transcription factors (e.g. 1N and XQ proteins) ; 2) modification of RNA secondary
structures by ribosomes or specific trans-acting factors (e. g. rho-mediated termination) ;
and 3) direct binding of a trans-acting factor to DNA serving as a steric impediment to
polymerase progress (e.g. lac repressor and Lex A protein) (reviewed by Spencer and
Groudine, 1990; Greenblatt et al., 1993).

NusA protein stabilizes the elongation conformation of RNAP. NusA enhances
pausing of RNAP at certain sites and is important for transcription termination at other
sites. NusA also serves to couple certain bacteriophage anti-termination factors (2. N
protein) to RNA polymerase when the operon contains an appropriate recognition
element (a nut site in the case of A N protein). Other E. coli elongation factors, NusB,
NusE and NusG, also modulate termination and antitermination.

Chamberlin and coworkers observed hydrolytic cleavage of the RNA by RNAP
near the 3' end of nascent transcripts (Surratt et al., 1991). For some arrested elongation
complexes, hydrolytic cleavage may be necessary to resume elongation. GreA and GreB
proteins promote RNA cleavage. GreB can be added to complexes after RNAP has
become arrested. GreA must be added before the complexes reach the arrest site to
promote hydrolysis (Borukhov et al., 1992; Borukhov et al., 1993). GreA recycles
between RN AP molecules and helps RN AP read through transcriptional pause sites. The
functions of GreA and GreB are very analogous to those of eukaryotic elongation factor
TFIIS.

In summary, accurate promoter recognition, efficient elongation, and regulated
termination require proteins that bind to the core catalytic component of RNAP. All
these proteins are RNA polymerase associating proteins (RAPs) in prokaryotes. Studies
of these factors provide models for understanding the mechanism and regulation of

eukaryotic RN AP 11.

Transcription by RNAP II

The constellation of accessory factors for accurate and regulated initiation by
RNAP II is more complex than that for bacterial RNAP. These factors have been
classified into four different categories (Matsui et al., 1980; Davidson et al., 1983;
Samuels et al., 1982; reviewed by Drapkin et al., 1993). The first category consists of
basal or general transcription factors, that specify accurate initiation and support a basal
level of initiation by RNAP H (reviewed by Weinmann, 1992; Zawel and Reinberg, 1993;
Conaway and Conaway, 1993; Buratowaki, 1994). The second group contains sequence-
specific regulators including activators and repressors. These factors have DNA binding
domains to recognize specific DNA sequences within promoters, enhancers, and
silencers, and regulatory domains to activate or repress transcription through direct or
indirect interaction with basal factors (reviewed by Ham et al., 1992; Tjian and Maniatis,
1994). These sequence-specific factors can influence initiation, elongation and
termination of transcription (Connelly and Manley, 1989a; Yankulov et al., 1994). The
third group termed adaptors, co-activators or mediators act as bridges between sequence-
specific transcription factors (activators and repressors) and basal factors to relay
regulatory signals to RNAP H (Dynlacht et al., 1991; Tanese et al., 1991; Merino et al.,
1993; Auble and Hahn, 1993; Ge and Roeder, 1994; Kretzschmar et al., 1994). The
fourth group consists of factors that influence elongation (Reinberg and Roeder, 1987;
Flores et al., 1989; Reines et al., 1989; Bengal et al., 1991; Marshall and Price, 1992).
Elongation factors may also be targets of activators or repressors and may interact with
mdiators to receive signals from regulators.

In order to initiate an RNA chain from a promoter DNA sequence accurately,
RNA polymerase H requires general transcription factors (TFHA, TFHB TFHD, TFHB,
TFIIF, and TFHH). Physiological elongation rates require additional factors (TFHS and

TFHX). Activators and repressors can potentially interact with the basal machinery to

6
dictate the level of transcription during initiation and/or elongation. Potentially,

termination and RNAP H recycling could be additional targets for transcriptional control.
Because of the complexity of the transcription cycle, regulatory checkpoints may include:
1) promoter recognition; 2) assembly of a preinitiation complex; 3) open complex
formation; 4) synthesis of the first phosphodiester bond; 5) transition from abortive
initiation to productive elongation; 6) formation of a stable ternary complex; 7)
elongation; 8) termination; and 9) recycling of polymerase.
General Transcription Factors

TFIID has a central function in transcription by RNAP H. The native TFIID is a
multiprotein complex consisting of the TATA-box-binding protein (TBP) and at least 7
tightly bound proteins termed TBP-associated factors (TAFns) in human (Pugh and
Tjian, 1991; Zhou et al., 1993), 8 TAFns in Drosophila (Dynlacht et al., 1991) and 9
TAFns in yeast (Reese et al., 1994). Biological and genetic evidence suggests that TBP
participates in RNAP I, RNAP H and RNAP IH transcription both in yeast and human
(Dahlberg and Lund, 1991; Simmen et al., 1991; Lobo et al., 1991; Comai et al., 1992;
Schultz et al., 1992; Sharp, 1992; Rigby, 1993; Hernandez, 1993). The SL1 complex for
RNAP I transcription consists of TBP, TAF1110, TAF163 and TAF148 (Comai et al.,
1994). The TFIHB complex for RNAP III transcription consists of TBP, TAFm70
('IDS4/BRF) and TAF11190/ 170 (Kassavetis et al., 1992; Taggart et al., 1992; Poon et al.,
1994). The human RNAP H TFIID complex consists of TBP, TAFHs of 250, 125, 95,
78, 50, 30 and 28 kDa (Zhou et al., 1993). Genes encoding most of the TAFs have been
isolated (Dynlacht et al., 1993; Goodrich et al., 1993; Hisatake et al., 1993; Hoey et al.,
1993; Kokubo et al., 1993, 1994; Ruppert et al., 1993; Weinzierl et al., 1993; Yokomori
et al., 1993a,b). TAFns have been suggested to stabilize binding of TBP to the carboxy
terminal domain (CI'D) of RN AP H (Conaway et al., 1992) and TFHB (Goodrich et al.,

1993). TAFs may also mediate signals from sequence-specific factors (Pugh and Tjian,

7
1990; Dynlacht et al., 1991; Hoey et al., 1993; Gill et al., 1994; Goodrich et al., 1993;

Serizawa et al., 1994a: Wang and Tjian, 1994; Xiao et al., 1994; Thut et al., 1995).

TBP is the only general transcription factor shown to have sequence-specific
DNA-binding activity. Recombinant TBP can form a stable complex with DNA
containing a TATA box. The size of TBP varies among species (yeast 27 kDa,
Arabidopsis 22 kDa, Drosophila 39 kDa and human 38 kDa), because the N-terminal
domain is highly divergent across species. The C-terminal 180 amino acids, however, are
highly conserved (reviewed by Greenblatt, 1991a). Minor differences within the
conserved C—terminal domain contribute to the species specificity of function rather than
the highly divergent N-terminal sequence (Cormack et al., 1991; Gill and Tjian, 1991).
Mutagenic study of TBP indicates that the conserved C-terminal domain can bind to the
TATA box and support basal transcription (Horikoshi et al., 1990; Hoey et al., 1990;
Peterson et al., 1990), and the divergent N-terminal domain may interact with co-
activators (Pugh and Tjian, 1990). Even though human TBP fails to replace yeast TBP
functionally in viva, human TBP and yeast TBP can substitute for each other in basal
transcription in vitro (Cormack et al., 1991; Gill and Tjian, 1991). TBP binds to the
minor groove of DNA (Starr and Hawley, 1991; Lee et al., 1991). The concave inner side
of TBP interacts with DNA via a curved antiparallel B-sheet, whereas the convex outer
surface can interact with many different proteins. Binding of TBP to promoter DNA
induces a 100° bend in DNA observed by a gel mobility shift assay (Horikoshi et al.,
1992) and by X-ray crystallography of a DNA/protein complex (Kim et al., 1993; Kim et
al., 1993). Bending may bring regulatory DNA sequences closer to the promoter. TBP
partially unwinds the DNA in the TATA box, but flanking sequences remain B-form
DNA. In order to compensate for unwinding, the DNA assumes a partial superhelical
twist. The biological function of this helix distortion is unknown.

TFHA has been identified as a heterodimer of 32 and 13.5 kDa subunits in yeast
(Ranish and Hahn, 1991) and a heterotrimer consisting of 34, 19, and 14 kDa subunits in

8
human (Ma et al., 1993; DeJong and Roeder, 1993). Yeast and human TFHA function

interchangeably in basal transcription (Ranish et al., 1992). Studies show that TFIIA
functions at an early stage of pre-initiation complex formation (Reinberg et al., 1987;
Flores et al., 1992). The binding of TFIID to DNA is stimulated by TFIIA through direct
interaction with TBP (Buratowski et al., 1989; Maldonado et al., 1990; Lee et al., 1992;
Yamamoto et al., 1992; Buratowski and Zhou, 1992). However, the TBP mutants that
cannot interact with TFHA, s'till perform basal transcription (Yamamoto et al., 1992;
Buratowski and Zhou, 1992). Also in a reconstituted transcription system, TFHA
becomes dispensable for accurate transcription when TFIID is replaced by recombinant
TBP (Cortes et al., 1992). Although TFHA is not necessarily required for basal
transcription, this factor may have an important role in activation and anti-repression
processes through interactions with TAF co-activators. TFHA has been suggested to
overcome inhibitory effects of a negative regulator of basal transcription named Dr2
(Merino et al., 1993; Ma et al., 1993; Liberman and Berk, 1994; Yokomori et al., 1994;
Ozer et al., 1994; Sun et al., 1994). TFHA seems to have an essential role in more
complex and regulated systems and becomes dispensable in highly purified systems.
TFHB is a single polypeptide of 33 kDa in human (Ha et al., 1991), 34.5 kDa in
Drosophila (Wampler and Kadonaga, 1992), and 35 kDa in yeast (Pinto et al., 1992). It
has been characterized as having multiple functional domains which can directly interact
with TBP, TAF1140, RNAP H, RAP30, and acidic activators (Lin and Green, 1991; Lin et
al., 1991; Tschochner et al., 1992; Goodrich et al., 1993; Roberts et al., 1993; Ha et al.,
1993; Barberis et al., 1993; Buratowski and Zhou, 1993). These biochemical studies
demonstrate that the N-terminus of RAP30 and HB interact with each other directly and
the C-terminal domain of TFHB interacts with TBP and also RNAP H. Also TFHB is
critical to interactions between the initiation complex and upstream activators.
Association of TFHB with the pre-initiation complex, in some cases, may be the rate-

limiting step in initiation. A genetic approach applied by Hampsey and coworkers shows

9
that mutations in yeast TFHB (Sua7p) cause a shift in transcriptional start site selection in

viva (Pinto et al., 1992; 1994). Mutations in the largest subunit (Sua8p) of yeast RNAP H
similarly alter initiation, suggesting that start site selection may involve interaction
between the largest RNAP H subunit and TFHB (Berroteran et al., 1994). The sua7 and
sua8 mutations cause initiation from new start sites but maintain initiation from normal
sites (Berroteran et al., 1994). These studies suggest that TFHB plays critical roles in
recruitment of RNAP II/TFIIF to the DA complex (a complex of promoter DNA, TFHA
and TFHD), communication to regulatory factors, and transcription start site selection.
TFHF (RAP30/74) in humans consists of two subunits performing important
functions in initiation, promoter escape, elongation, and polymerase recycling (Burton et
al., 1986, 1987, 1988; Chang et al., 1993; Reinberg and Roeder, 1987; Flores et al., 1989;
Bengal et al., 1991; Izban and Luse, 1992a). Recent work also indicates that TFHF can
be a target for transcriptional activators (Zhu et al., 1994; Joliot et al., 1995). RAP30 and
RAP74 subunits can perform separable functions in initiation and very early elongation
(see Chapter H; Chang et al., 1993). The apparent molecular weight of TFIIF is 220-280
kDa as determined by gel filtration studies, suggesting a heterotetrameric structure
(Conaway and Conaway, 1989; Flores et al., 1990; Kitajima et al., 1990; Wang et al.,
1994). TFHF subunits were initially identified as polypeptides capable of binding to
RNAP H (Sopta et al., 1985). Both subunits of TFHF are essential for accurate
transcription in HeLa-cell nuclear extracts, from which TFIIF was removed by
immunoprecipitation with antibody directed against RAP30 (Burton et al., 1986, 1988).
Anti-RAP30 antibody co-precipitates RAP74, indicating that RAP30 and RAP74 are
normally associated in a complex (Burton et al., 1988). Binding to RNAP H prevents
phosphorylation of RAP30 but not RAP74, suggesting that the RAP30 subunit interacts
directly with RNAP H (McCracken and Greenblatt, 1991). However, both recombinant
RAP30 and RAP74 can bind to RNAP H in a gel-mobility shift assay independently
(Killeen and Greenblatt, 1992; Tyree et al., 1993; Wang and Burton, 1995). RAP74

10
inhibits transcription of RNAP H in a non-promoter transcription assay in the absence of

RAP30, and the C-terminal region of RAP74 binds tightly to RNAP H (Wang and
Burton, 1995). TFIIF inhibits nonspecific binding of RNAP H to free DNA (Conaway
and Conaway, 1990; Killeen and Greenblatt, 1992) and RAP30 assists RNAP H to bind
to the DAB complex (Flores et al., 1991; Killeen et al., 1992). However, recombinant
RAP30 itself cannot release RN AP H from non promoter DNA once it has bound,
although TFHF has this capacity (Killeen et al., 1992; Killeen and Greenblatt, 1992).

TFIIF interacts with TFHB through the RAP30 subunit (Ha et al., 1993), and also
with serum response factor through the RAP74 subunit (Zhu et al., 1994; Joliot et al.,
1995). Gel mobility shift assays suggest that RAP30/RNAP H can bind to the DAB
complex (TFHD TFIIA TFIIB and promoter DNA). In gel shifts, the intensity of the
DABPolF complex is dramatically increased by RAP74 (Flores et al., 1991). Therefore,
RAP74 strongly increases the stability of polymerase association with the promoter,
although this TFHF subunit is not required for polymerase entry.

TFHF isolated from human (RAP30/74), rat (By), and fly (Factor 5) contains two
subunits, but the counterpart in yeast (factor g) consists of three subunits, nglp/Ssu71p,
ng2p, and ng3p/Anc1p/TAF30 (Henry et al., 1992; 1994). Studies of S. cerevisiae
demonstrate that the yeast counterpart of RAP74 (Ssu7lp) interacts with TFHB (Sua7p)
genetically (Sun and Hampsey, submitted). Mutations in RAP74(Ssu71p) suppress both
a cold-sensitive growth phenotype and the altered initiation pattern conferred by a TFHB
defect. The RAP30 counterpart in yeast is ng2p. From recent studies of RNAP H
binding proteins in yeast, Cdc73p appears to be have a function related to those of ng2p
and RAP30 (See Chapter IV of this thesis; Wade et al., 1995; Fang and Burton, 1995).
Cdc73p may be part of an alternate TFHF function in yeast. ng3p/Anclp functions in
stimulating transcriptional activity and can be dispensable for basal transcription (Henry
et al., 1992). ng3p/Anclp has been shown to be TAF30 of the yeast TFIID complex
(Henry et al., 1994). ng3p/Anclp has also been identified as the 30 kDa subunit of the

1 1
SWI/SNF complex, which alters chromatin structure to activate transcription (Cairns et

al., 1994).

Since TFHF is involved in many phases of the transcription cycle, regulators
interacting with TFHF might impact on promoter recognition, transcription complex
assembly, initiation of RNA synthesis, promoter escape, and elongation.

TFIIE is a heterotetramer composed of two 34- and 56-kDa subunits (Ohkuma et
al., 1990; Inostroza et al., 1991), that can stably associate with RNAP H in solution.
TFHB has been purified as one of the RAPs from an RNAP II affinity column
(Buratowski et al., 1991; Flores et al., 1989). TFIIE binds to the form of RNAP H that is
not phosphorylated on the CTD (RNAP IIA) through the 56—kDa TFIIE subunit (Maxon
et al., 1994). TFIIE stimulates the CT D kinase activity of mm promoting CTD
phosphorylation (Serizawa et al., 1994b; Ohkuma and Roeder, 1994). TFHB has also
been found to modulate the helicase activity of TFIIH either negatively (Drapkin et al.,
1994a) or positively (Serizawa et al., 1994b) through a direct interaction with the largest
subunit of TFIIH. It has been suggested that TFIIE assembly during pre-initiation
complex formation is after assembly of RN AP II/TFIIF but before subsequent
recruitment of TFIIH (Flores et al., 1992), and indeed TFIIE interacts with TFIIH and
both subunits of TFIIF (Maxon et al., 1994). Recent studies indicate that TFHE along
with ‘1le and ATP are not required for initiation, but instead are required for promoter
escape, suggesting that they are necessary for conversion of an initiated complex to an
elongation complex (Goodrich and Tjian, 1994).

TFHH is a multisubunit transcription factor with polypeptides 34, 38, 41, 44, 50,
62, 80, and 89 kDa (Schaeffer et al., 1993; 1994). TFIIH has two catalytic activities, an
ATPase/helicase activity involved in promoter escape (Goodrich and Tjian, 1994), and a
kinase activity involved in CTD phosphorylation of RNAP H (Lu et al., 1992). TFIIH
also phosphorylates TBP, the 56 kDa subunit of TFIIE and the RAP74 subunit of TFHF
(Ohkuma and Roeder, 1994). The p62 subunit of TFIH-l interacts with the activation

12
domains of VP16 and p53 indicating that TFIH-I is a target of regulators (Xiao et al.,

1994). Transcriptional activity and CTD kinase co—fractionate, and anti-p62 antibody
inhibits both transcriptional and kinase activities. None of the TFIIH subunits, however,
has been shown to have intrinsic CTD kinase activity. The largest and the second largest
subunits of TFHH, p89 and p80, in human contain consensus ATPase/helicase motifs and
have been shown to be identical to the XPB-ERCC3 and XPD-ERCC2 excision repair
proteins (Schaeffer et al., 1993; Feaver et al., 1993). Therefore, TFIIH functions in both
transcription and DNA repair. The yeast counterparts of these helicases (SSL2 and
RAD3) are both essential for cell viability and transcriptional activity (reviewed by
Drapkin and Reinberg, 1994a; 1994b); however, point mutations in the nucleotide
binding domain of the second largest subunit (RAD3) are not lethal and result only in
defects to nucleatide-excision repair (Feaver et al., 1993; Sung et al., 1988), whereas
similar mutations in the largest subunit (SSL2) are lethal. Extracts derived from 3le
mutants grown under the non-permissive condition are transcriptionally inactive (Guzder
et al., 1994). Recent evidence indicates that there are two forms of TFIH-I, a halo-TFIIH
involved in transcription and a repairasome responding in nucleotide-excision repair
(Svesjstrup et al., 1995). Thus, TFIHi appears to play a critical role in ATP hydrolysis
and open complex formation, CTD phosphorylation, promoter escape, and elongation of
RNAP H when template DNA contains a damaged base.
RNA polymerase H and RAPs

RNA polymerase H (RNAP II) is the nuclear DNA—dependent RNA polymerase
that synthesizes mRNA in eukaryotic cells. Several conserved features exist among
RNAP H isolated from different species. RNAP 11 contains 10-12 subunits and the
relevant subunit sequences are highly conserved between species. RNAP H has two large
subunits homologous to the largest subunits of RNAP I, RNAP HI, and the [3' and B
subunits in prokaryotes. The largest subunit of RNAP H contains a unique C-terminal

domain (CTD), not present in other polymerases, composed of multiple heptapeptide

13
repeats with the consensus sequence YSPTSPS, 52 repeats in the mammalian enzyme.

The largest RNAP H subunit contains a DNA binding site and the second largest subunit
binds nucleotide substrates. Both subunits contribute to the active site for RNA
synthesis. Finally, RNAP H contains three common subunits of 14-28 kDa also found in
RNAP I and RNAP 1H, and other small subunits unique to RN AP H (reviewed by Young,
1991).

The CT D is multiply phosphorylated on the two SP serines within each
heptapeptide repeat by a stably associated CTD kinase using either ATP or GTP as
substrate. The highly phosphorylated form of RNAP H is referred to as the H0 form, and
the dephosphorylated form as the IIA form (Cadena and Dahmus, 1987; Kim and
Dahmus, 1989).

RNAP H binds to the DB complex in the RNAP HA form. The dephosphorylated
CTD binds to the TBP subunit of TFIID. Conversion of the CTD to the phosphorylated
form in the pre-initiation complex reduces the affinity of the CTD for TBP (Dahmus and
Kedinger, 1983; Layboum and Dahmus, 1989; Usheva et al., 1992). Transition from a
pre-initiation complex to an elongation complex, therefore, involves CT D
phosphorylation and RNAP 110 is the primary elongation form of polymerase.
Phosphorylation of the CTD, however, is not the only requirement for the transition from
the preinitiation complex to the initiated complex. RNAP H from which the CTD has
been removed (termed the HB form) can accurately initiate in an ATP-dependent reaction
(Zehring et al., 1988). Moreover, transcription initiation can be uncoupled from CTD
phosphorylation using a protein kinase inhibitor (Serizawa et al., 1993). Therefore,
Conaway and coworkers suggest that phosphorylation of the CTD is not an essential step
in basal transcription or factors that establish the requirement for CTD phosphorylation
are missing in some in vitro systems (Serizawa et al., 1993). Although phosphorylation
of the CTD and the CTD itself are not required for the catalytic activity of RNAP H in
vitro, the CTD is essential in viva (Nonet et al., 1987; Allison et al., 1988; Zehring et al.,

14
1988; Bartolomei et al., 1988). Genetic and biochemical evidence suggests that the CTD

plays a role in mediating transcription activation by upstream regulators (Allison and
Ingles, 1989; Usheva et al., 1992). Chromosome fluorescence staining using antibody
against the hyperphosphorylated or hypophosphorylated CTD suggests that polymerase is
in the HA form in stalled elongation complexes and 110 form in active elongation
complex (Weeks et al., 1993; O'Brien et al., 1994).

RAP is an acronym for an RNA polymerase H associating protein, and these
factors have been isolated using affinity methods in which RNAP II is immobilized along
with bound factors (Sopta et al., 1985; Burton et al., 1986; Greenblatt, 1991b; Wade et
al., 1995). RAPs have been identified as initiation factors (e.g. TFHF (RAP30/74), TBP,
TFHB, TFIIE, TFHH), elongation factors (e.g. RAP30/’74, RAP38), and possible
mediators (e.g. AF9/ENL, Anclp). RAPs may bind directly or indirectly to RNAP H.
Such factors are not identified as RNAP II subunits because of dissociation during
purification, just as the E. cali sigma factor and NusA can be separated from "core"
RNAP (Burgess et al., 1969; Greenblatt and Li, 1981). RAPs have generally been
considered accessory factors rather than subunits of RNAP H because binding is sensitive
to elevated salt concentration (0.3-0.5 M KCl) and in many cases RAPs can be exchanged
from one RNAP H to anorher. Dissociable interaction between RAPs and RNAP H
allows regulatory mechanisms in which these contacts are modified. TFIIF and TFIIH,
for instance, appear to be targets for transcriptional regulators. RAP activity can also be
controlled by phosphorylation or other covalent modification. Recently, holo enzyme
forms of RNAP H have been isolated from yeast (Koleske and Young, 1994) or partially
reconstituted using mammalian factors (Conaway et al., 1992). Such higher order
assembly complexes can accurately initiate and respond to regulatory signals with
minimal supplementation of factors.

TFIIF subunits RAP30/74 were first isolated using an RNAP II affrnity column
(Sopta et al., 1985; Burton et al., 1986). Recently, yeast RAPs were isolated using an

15
anti-CTD monoclonal antibody immobilized on a column to capture RNAP II and

associated factors (Wade et al., 1995). This direct capture protocol may allow isolation
of RAPs not detected using the original methods, because exchange of factors to
immobilized RNAP H is not required.

Some transcription factors that bind RNAP H through the CTD might be lost by
anti-CTD chromatography. This appears to be the case for TBP, TFIIH, and suppressor
of RNA polymerase B proteins (Srbps) (Fisher et al., 1992; Koleske et al., 1992;
Thompson et al., 1993). The yeast RAPs isolated from an anti-CTD column included
known transcription factors TFHB, TFIIF and TFIIS, as well as several proteins of
unascribed transcriptional functions. As discussed above, yeast TFIIF consists of three
subunits namely Ssu7 1p fl‘fglp, ng2p and Anclp/ng3p. Most interestingly, a possible
RAP30 homologue named Cdc7 3p was identified by protein sequence analysis (Wade et
al., 1995).

In prokaryotes, RNA polymerase obtains its promoter specificity by contacting
with different 0 factors. The polymerase binding domains of human RAP30, yeast
ng2p, and Cdc73p, and bacterial sigma factors show sequence similarity and conserved
function (Wade et al., 1995; Fang and Burton, 1995). Limited homology to bacterial 0
factor DNA binding domain has also been found in the fourth subunit of yeast RNAP H
and mitochondrial RNA polymerase (reviewed by Jaehning,1991). The CDC7 3 gene was
isolated as a suppressor of a deletion mutant in the STE2 gene which encodes the a-factor
receptor. The cdc73-1 mutant enables the ste2 deletion mutant to mate at permissive
temperature and arrests the cell cycle in GI upon temperature upshift (Reed et al., 1988).
Our work confirms that Cdc73p is a yeast RAP and is similar in sequence to human
RAP30 (Wade et al., 1995; Chapter IV). Therefore, Cdc73p can potentially function to
link the regulation of cell cycle and transcription. Cdc73p activity could be controlled by

cell cycle signal(s) and may function as an alternative RAP30 in transcription of specific

16
genes, in much the same way that alternate o-factors are responsible for transcription
from alternate promoters in bacteria.

Another yeast RAP Anclp/ng3p may function as a mediator or co-activator
rather than as a basal factor. Anclp is not essential for yeast viability or for accurate
transcription, although transcription is stimulated by this factor (Henry et al., 1992). In
addition to our identification of Anclp as a RAP (Wade et al., 1995), Anclp is also a
subunit of TFIIF, a yeast TAF (Henry et al., 1994) and a component of the SWI/SNF
complex (Cairns et al., 1994). SWI/SNF alters nucleosome structure and can activate
transcription on nucleosomal DNA (Imbalzano et al., 1994).

Mechanism Of RNA Polymerase H Transcription

RNAP H and basal factors c00perate to form a complex with capacity to bind a
promoter specifically and to initiate transcription accurately. This elaborate process
involves multiple steps including: 1) template activation; 2) promoter recognition; 3)
DNA strand separation; 4) abortive initiation; 5) formation of a stable ternary complex; 6)
processive elongation; 7) termination of transcription and 8) cycling of RNAP H. The
mechanism of each phase is discussed as follows.

1) Template Activation

Before a promoter can be recognized, chromosome structure must be altered to
expose the DNA sequence. The alteration from an inactive template to an active one
must be influenced by cellular factors. Studies on histone-mediated transcription
repression indicate that formation of active transcription complexes on a promoter
competes directly with the assembly of the DNA into nucleosomes (Workman et al.,
1990; reviewed by Paranjape et al., 1994). Activators enhance transcription in two ways:
1) a process referred to as antirepression which relieves chromatin-mediated repression of
transcription (Croston et al., 1991), and 2) a true activating process which facilitates
formation of the transcription complex (Layboum and Kadonaga, 1992).

2) Promoter Recognition

17
Accurate transcription is initiated from promoter sequences (Weil et al., 1979;

Manley et al., 1980). Two working models have been established for preinitiation
complex formation, namely a multiple-step assembly pathway (reviewed by Zawel and
Reinberg, 1993) and a holoenzyme RN AP 11 complex pathway (reviewed by Serizawa et
al., 1994; Koleske and Young, 1995).

The core promoter (minimal DNA sequence required for basal transcription) can
be roughly classified into two categories: 1) a TATA-containing promoter; and 2) a
TATA-less promoter. On a TATA-containing promoter, the interaction between DNA
and TBP serves as a foundation for transcription complex assembly. Transcription of
RNAP H on some promoters without the consensus TATAAA sequence still requires
TFHD to function. The recruitment of TBP to a TATA-less promoter requires tethering
to upstream activators (e.g. SP1 and TAF interaction) and/or an initiator (Inr) binding
protein (e.g. TFHI and TAF interaction). Inr is the DNA sequence encompassing the
transcription initiation site. Now it appears that most promoters contain an Inr, although
the nucleotide sequence of this element is not highly conserved (reviewed by Weis and
Reinberg, 1992). Several lines of evidence also suggest that TBP can bind to the -30
region relative to the transcription start site on a TATA-less promoter (Wiley et al., 1992;
Zenzie-Gregory et al., 1993). In any case, either TATA or Inr motif alone can potentially
direct transcription initiation (Myers et al., 1986; Smale et al., 1990; Nakatani et al.,
1990). When these two motifs are present together, they can function cooperatively
(Smale and Baltimore, 1989; Nakatani et al., 1990; Conaway et al., 1990).
(BMW

In the ordered-assembly model (Figure 1), TATA-binding protein (TBP) first
binds to the TATA motif of the promoter. The binding of TFHD to DNA is facilitated by
TFIIA, and under certain conditions, TFIIA is not absolutely required for the
transcriptional activity. TFIIB then binds to form a DAB complex (a complex consisting

of TFHD, TFIIA, TFHB and promoter DNA) (Buratowski et al., 1989; Maldonado et al.,

18
1990). RNA polymerase 11 associated with RAP30/74 (TFIIF) binds the DAB complex.

TFHF binding to RNAP II suppresses non-specific DNA binding by polymerase and
promotes stable association of RNAP H with the promoter. Although both RAP30 and
RAP74 associate with polymerase in viva, RAP30 is necessary and sufficient to guide
polymerase to the DAB complex (Killen and Greenblatt, 1992; Flores et al., 1991). The
RAP74 subunit stabilizes this interaction. Consistent with these observations, our study
demonstrates that RAP74 can function at a later stage of the transcription process than
RAP30 (Chang et al., 1993; Chapter H). After formation of the DBPolF complex, TFHB
and TFHH join and form the DABpolFEH complex. On linear DNA templates, TFHB
and TFIH-I are necessary to fully convert the closed DNA complex to an open,
transcriptionally competent complex. It has been demonstrated that activators can
interact with several basal transcription factors; therefore, the binding affinity of proteins
in each assembly step could be modulated by protein-protein interaction between
regulators and basal factors.
(II) Wm

Recent evidence suggests that aggregates of basal and regulatory factors can
assemble on RN AP 11 to constitute holoenzyme forms. RN AP II holoenzymes
mayperform specific functions within cells, such as regulated initiation or elongation of
RNA chains. Holoenzyme forms have been isolated or reconstituted that can bind to the
promoter with minimal supplementation of additional factors (Conaway et al., 1992;
Koleske and Young, 1994; Kim, Y.-J. et al., 1994). Some of these complexes maintain
their integrity during many steps of purification, and can support activated transcription.
Other aggregates can be dissociated under mild conditions, but may represent important
RNAP H forms that exist in vivo.

Many of the individual protein-protein contacts that contribute to holoenzyme
formation have been identified. For instance, TFHB interacts directly with TBP and
RNAP H to recruit RNAP II/I‘FIIF (Barberis et al., 1993; Buratowski and Zhou, 1993).

19

Figure 1. Preinitiation complex formation pathways: a multiple-step assembly pathway

and a holoenzyme assembly pathway.

    

RNAP II
Holo Enzyme

 

+1

 

20

 

 

 

 

 

 

Figure 1

21
The N-terminus of RAP30 and TFHB interact (Ha'et al., 1993). Furthermore, TBP and

TFHB can bind RNAP II through the CTD (U sheva et al., 1992; Maxon et al., 1994). Our
laboratory has observed a number of RAPs associating with RNAP H isolated as a
mixture of holoenzyme forms by anti-CTD affinity chromatography, including TFHB,
TFHF, TFHS, and Cdc73p (Wade et al., 1995).

Assembly mechanisms and holoenzyme forms indicate an essential role for TFIIF
in establishment and maintenance of complexes. Since RAP30 interacts with TFHB
directly (Ha et al., 1993), and RAP74 increases the stability of the RNAP II/TFIIF
complex (Garret et al., 1992), the overall stability of a holoenzyme likely depends on
TFIIF, placing this factor at the core of the assembly process. Since TFHF is part of both
initiating and elongating polymerase, it is likely an important scaffold for construction of
multiple holoenzyme forms for different stages of the transcription cycle.

Since the multi-step pathway was established according to the minimal contacts
necessary for assembly in vitro, holoenzyme forms are expected to more closely resemble
physiological forms of RN AP 11. It may be that on some promoters, RNAP H binds in a
particular holoenzyme form. On other promoters, particular contacts between factors
may be regulated, and a mixture of holoenzyme and assembly mechanisms may be
important for initiation. Elongatin g polymerase holoenzymes are likely to differ
substantially from initiating forms.

Interestingly, under some circumstances, RN AP H can accurately initiate in the
absence of a full complement of basal transcription factors. For instance, on supercoiled
templates, initiation can occur in the absence of TFHE and TFIH-I (Parvin and Sharp,
1993; Tyree et al., 1993). Negative supercoiling of the template removes the requirement
for DNA strand separation by TFIIH, and TFIIE participates in this process (Goodrich
and Tijan, 1994). In the case of a superhelical IgH promoter, TFIIF was dispensible for

accurate transcription (Parvin and Sharp, 1993). Surprisingly, on the adeno-associated

22
virus (AAV) P5 promoter, TFIID is dispensible for accurate initiation. In this case YYl,

an Int-binding protein, TFIIB and RN AP 11 are sufficient to direct basal transcription
from a supercoiled template (Usheva and Shenk, 1994).

Whether these minimal complexes have physiological relevance is not known. In
highly purified transcription systems, TBP can substitute for TFIID, but in mammalian
cell extracts TBP is assembled into TBP/T AF complexes. In the presence of TAFs, other
cooactivators and regulators, transcription is likely to require a more complete
constellation of factors. Some promoters, on the other hand, may have slightly different
requirements than those studied to date, and may initiate by different mechanisms using
an alternate collection of factors. In Chapter IV of this thesis, a yeast protein named
Cdc73p is identified that may be a component of an alternate form of TFIIF and may
support an alternate transcriptional mechanism by RN AP H.

3) Open complex formation
. DNA strands must be separated to expose the template for phosphodiester bond
synthesis. In higher eukaryotes, initiation occurs 25 to 30 nucleotides downstream from

the TATAAA sequence (Corden et al., 1980). DNA strand separation can be detected by
modification of single-stranded DNA with reagents such as KMnO4. Productive

initiation requires hydrolysis of ATP at the [3-7 bond position (Bunick et al., 1982;
Sawadogo and Roeder, 1984). This is a unique requirement of RNAP 11, since RNAP L
RNAP HI, and bacterial RNAP do not require ATP hydrolysis for initiation. RNAPs of
all sorts utilize ATP as a substrate for elongation, but as RNA chains are elongated, it is
the a—B bond of ATP that is hydrolyzed. ATP analogs such as AMPPNP can substitute
for ATP for initiation and elongation by most polymerases, but not for initiation by
RNAP H. Full start site Opening of the DNA helix requires ATP or ATP analogs with a
hydrolyzable B—q bond (Wang et al., 1992; Jiang and Gralla, 1995). At sufficiently high
concentrations, GTP, CTP and UTP can be substituted for ATP as the substrate for B-y
bond hydrolysis (Wang et al., 1992; J iang et al., 1994; Jiang and Gralla, 1995), but only

23
ATP has been shown to support this function for runoff transcription in vitro. Perhaps

ATP hydrolysis supports more than one requirement in initiation of RNA chains by
RNAP H.

A helicase activity has been proposed to carry out the DNA unwinding reaction in
RNAP H initiation (Burton et al., 1988). Two subunits of TFIH-I have been shown to
include ATP-dependent helicase activities (Feaver et al., 1993; Serizawa et al., 1993;
Schaeffer et al., 1993). Current evidence suggests that the largest TFIHI subunit is the
helicase required for transcription, and the Other helicase subunit is required for DNA
repair (Guzder et al., 1994). A template already in the open conformation can be
accurately transcribed without addition of ATP (Tantin and Carey, 1994). As mentioned
above, highly supercoiled templates do not require TFIIH for initiation, nor do they
require ATP hydrolysis.
4) Abortive initiation and promoter escape

RNAP 11, similar to prokaryotic RNA polymerase, goes through an abortive phase
of transcription in which short transcripts are synthesized and released before productive
elongation occurs (reviewed by McClure, 1985). An open binary complex can initiate
phosphodiester bond synthesis in the presence of all four nucleoside triphosphates.
However, these newly synthesized RNAs are unable to associate with polymerase as a
stable ternary complex until about 10 phosphodiester bonds have been formed (Chapter
IH; Chang and Burton, 1995). In our system this process requires about 15 s. Transcripts
5 to 10 nucleotides in length are not stably associated with polymerase and are released as
abortive transcripts (Luse and Jacob, 1987; Luse, 1990; Jacob et al., 1991, 1994).
Promoter escape, also called promoter clearance, is the transition between abortive
initiation and productive elongation. One explanation for abortive initiation is that
polymerase must overcome strong protein-protein and protein-DNA contacts holding it to
the promoter. The molecular forces that drive promoter recognition and accurate

initiation are in competition with those that drive elongation. Polymerase must make a

24
transition from being a sequence-specific DNA binding protein, with high selectivity for

promoter sites, to an elongation enzyme that has little ability to discriminate between
different sequences. Presumably, this transition requires conformational changes in
polymerase and release Of some initiation factors. For instance, escape from abortive
initiation in bacterial systems involves the release Of sigma factor and significant changes
in polymerase conformation. Template contacts also change dramatically as promoter
escape occurs (Straney and Crothers, 1987; Krummel and Chamberlin, 1989; Metzger et
al., 1989).

The RAP74 subunit of TFHF has important functions in promoter escape by
RNAP H (Chapter II; Chang et al., 1993). Analysis Of short transcripts formed in the
presence of RAP74 mutants demonstrates that the stability Of short ternary complexes is
dependent on RAP74 (Chapter HI). It is likely that the function of RAP74 in promoter
escape relates to stabilization of short transcripts.

The helicase function of TFIIH has been proposed to drive promoter escape
(Goodrich and Tjian, 1994; Maxon et al., 1994). Phosphorylation of the CTD reduces the
affinity Of RNAP II for TBP, and may be another feature of the transition between
initiating and elongating enzyme (Usheva et al., 1992; Layboum and Dahmus, 1989).

Interestingly, abortive initiation does not require TFHH or ATP hydrolysis,
although an accessible template strand is required (Goodrich and Tijan, 1994; Timmer,
1994). When open complex formation is monitored with the single-strand cleavage
reagent O-phenanthroline-copper, TFIIH and ATP hydrolysis are found to be not required
for sensitivity of the DNA. Apparently, the Open complex is formed in two steps. The
initial step requires assembly of the DABpolF complex. The Open complex formed under
this condition will support abortive initiation but not promoter escape and productive
elongation. This open complex can be detected with O-phenanthroline-copper, but not
with KMnO4. To free polymerase from the promoter requires an ATP-dependent step in

which the TFIIH transcription helicase makes a more extensive open complex.

25
Movement of TFIIH may be important to remodel protein-protein interactions that

otherwise hold RN AP H at the promoter.

Recently Chamberlin and coworkers demonstrated that RNAP H has two RNA
binding sites responsible for the association of RNA products with transcription complex
(Johnson and Chamberlin, 1994; Chamberlin, 1995). Similar RNA binding sites have
also been observed in E cali RNAP (Borukhov et al., 1993; Mustaev et al., 1994). A low
affinity site is located at the catalytic center of the enzyme where phosphodiester bonds
are synthesized and requires a chain length greater than about 10 nucleotides for
occupancy. A higher affinity site is located 10-12 nucleotides distant from the catalytic
center. Instability of short RNAs, therefore, may relate to the ability to fill in the low
affinity site. Transcripts Of less than about 10 nucleotides are weakly bound and subject
to release as abortive transcripts.

5) Elongation

As each new phosphodiester bond is formed, polymerase makes a molecular
decision whether to elongate or to terminate the chain. Polymerase can pause, and after
traversing some kinetic barrier, resume elongation. Arrested complexes are distinct from
paused complexes. These are stalled complexes that cannot be elongated without some
event, such as nucleolytic cleavage of the transcript, to re-establish the association of the
catalytic center with the template. Elongating, paused, arrested, and terminating
complexes are characterized by binding Of the transcript to polymerase, RNAP H
conformation, and DNA contacts. Factors that regulate elongation and termination are
expected to affect these processes through interactions with polymerase, transcript, and
template (reviewed by Spencer and Groudine, 1990; Kerppola and Kane, 1991; Krumm et
al., 1993; Greenblatt et al., 1993; Kane, 1994). .

Pausing is thought to be mediated through interaction with specific sequences
within DNA or RNA. Pause sites can be recognized by RNAP H in the absence Of other
factors (Reines et al., 1987; Kerppola and Kane, 1988; Wiest et al., 1992). Differential

26
modification of the polymerase CTD by phosphorylation may alter interaction with pause

sequences. Elongation factors TFIIF, TFIIS, and TFIIX increase RNAP H elongation
efficiency perhaps by suppressing pausing (Reinberg and Roeder, 1987; Flores et al.,
1989; Reines et al., 1989; Bengal et al., 1991; Izban and Luse, 1992a; Yankulov et al.,
1994). These factors have distinct activities that depend on whether they are added
before or after RNAP H reaches the pause site. TFHF and TFIIX stimulate the rate of
elongation and promote reading through pause sites. TFIIS exerts its influence on
transcription after polymerase has paused.

To recover arrested complexes, endonucleolytic cleavage of the nascent transcript
is necessary (Reines et al., 1992; Izban and Luse, 1992b, 1993; Wang and Hawley, 1993;
Rudd et al., 1994; reviewed by Reines, 1994). Two distinct cleavage reactions have been
identified. Pyrophosphorolysis is the reverse of the normal polymerization reaction, and
hydrolytic cleavage of the transcript near the 3' end can also occur. RNAP H will
catalyze these reactions in the absence of elongation factors indicating that these catalytic
processes are intrinsic to polymerase. TFIIF stimulates the pyrophosphorolysis reaction,
and TFIIS stimulates the rate of transcript hydrolysis (Reines et al., 1992; Wang and
Hawley, 1993; Rudd et al., 1994).

Human immunodeficiency viruses (HIV) transacting protein Tat is a potent
activator of transcription from the HIV-1 long terminal repeat (LTR) promoter (reviewed
by Sharp and Marciniak, 1989; Cullen, 1990, 1993; Greenblatt et al., 1993). Tat
stimulates initiation (Laspia et al., 1989; Southgate and Green, 1991), elongation, and
processivity (Marciniak and Sharp, 1989; Laspia et al., 1989, 1990; Marciniak et al.,
1990; Kato et al., 1992; Zhou and Sharp, 1995). Tat functions through binding to an
RNA element termed TAR (Muesing et al., 1987; Hauber and Cullen, 1988). Roeder and
coworkers Observed that TFIIF increases the basal level of elongation but not Tat-
activated stimulation, whereas TFIIS shows synergistic stimulation Of elongation with

Tat. Antiserum directed against the RAP74 subunit of TFIIF preferentially suppresses

27
the activated level of transcription exerted by Tat (Kato et al., 1992). These observations

lead the authors to suggest that Tat may stimulate elongation through RAP74. Landick
and coworkers Observed that Tat augments the effect of TFHF on RNAP H processivity
and suggested that stimulation Of transcription elongation by Tat occurs at least partially
by recruitment Of TFIIF to the elongating transcription complex(Meier et al., 1994).
However, a direct protein-protein interaction between Tat and TFHF has not yet been
reported.

Price and coworkers reported that productive elongation complexes are derived
from early paused elongation complexes by the action of a factor P-TEF (positive
transcription elongation factor) to release an N-TEF (negative transcription elongation
factor) (Marshall and Price, 1992). RNAP II complexes that are not appropriately
modified are defective for processivity and terminate transcription. The identities of N-
and P-TEF, however, have not yet been reported.

In viva RN AP II molecules have been identified that are initiated but are stalled
for elongation near the 5' ends of various genes (Rougvie and Lis, 1990; Bengal et al.,
1991; Giardina et al., 1992; Krumm et al., 1992; Meulia et al., 1992; Strobl and Eick,
1992; Kash et al., 1993). For instance, on the hsp70 and hsp26 promoters Of Drosophila
melanogaster, RNAP II has been found to be initiated but stalled within the first 29
nucleotides of the mRN A chain prior to heat shock. After heat shock, polymerase
molecules are found distributed throughout the hsp70 gene, and the mature hsp70 mRNA
and protein are produced (Rougvie and Lis, 1990; Giardina et al., 1992). Even after the
hsp70 gene is activated by heat shock, the pause site for polymerase near the promoter is
maintained and the single-stranded DNA region at the pause site can be detected with
KMnO4. The implication of these Observations is that the heat shock transcription factor
(HSF) stimulates polymerase to traverse the pause site and productively elongate the

transcript through the gene, in addition to effects HSF may have on initiation. Pausing in

28
this case may poise RNAP H and the hsp70 transcription unit to respond quickly to the

heat shock signal.

The phosphorylation status Of the CTD of stalled RNAP H molecules on hsp70
and hsp26 genes has been examined before and after heat shock. Hyperphosphorylated or
nonphosphorylated RNAP H molecules were detected after ultraviolet cross linking,
using specific antibodies directed against the different polymerase forms (Weeks et al.,
1993). RNAP H in productive elongation complexes after heat shock has a
phosphorylated CT D, while the CTD Of the paused polymerase is mainly
unphosphorylated (O'Brien et al., 1994). Since the primary elongation form Of RNAP H
is hyperphosphorylated, this result is consistent with the presumed function of CTD
modification in the transition between initiating and elongating forms of polymerase. On
heat shock genes, phosphorylation of the CTD occurs after initiation to drive polymerase
into productive elongation.

6) Termination

The mechanism for RNA polymerase II to terminate a chain has not been clearly
elucidated. 3'-end formation Of mRN A transcripts involves transcript cleavage
downstream of an AAUAAA processing and polyadenylation signal. Polymerase appears
to lose processivity after processing occurs and to terminate at many sites beyond the
polyadenylation signal. Termination by RN AP H at the 3' end of genes encoding poly(A)
mRNAs is thought to require several distinct cis-acting elements including: 1) a
functional poly(A) signal (Connelly and Manley, 1988; Laniox and Acheson, 1988;
Logan et al., 1987; Whitelaw and Proudfoot, 1986); 2) a downstream transcriptional
pause site (Logan et al., 1987); 3) a structural element causing a bend in the DNA helix
(Kerppola and Kane, 1990); and 4) termination signal sites on the 5' flanking region of
promoters which can be specifically recognized by trans-acting factors (Connelly and
Manley, 1989a, 1989b; Meulia et al., 1992; Roberts et al., 1992). Perhaps CI‘D

phosphorylation is decreased after polymerase traverses a polyA addition site.

29
7) Recycling of RNAP II

Polymerase must be in the dephosphorylated state to efficiently associate with a
promoter. Since elongating polymerase is highly phosphorylated, a CI'D phosphatase is
likely to be important for polymerase recycling after termination. Chambers and Dhamus
recently reported isolation of a CTD phosphatase (Chambers and Dhamus, 1994). CTD
phosphatase functions by binding to a site on RNAP H other than the CTD and is strongly
stimulated by the RAP74 subunit of TFIIF (Chambers et al., 1995).

Response of the general mechanism to activators

Each phase of the transcription cycle is a potential target for regulation. Basal
factors are now thought to be required for initiation of all genes; activators and repressors
dictate the rate at which the basal complex initiates transcription. Initiation can be
stimulated by interaction between promoter-, silencer-, and enhancer-binding factors.
Activation domains of regulators, for instance, have been shown to interact directly with
TBP and TFIIB. In some cases, co-activators such as TAFs are important for
transmission of these signals. In an ordered assembly model, a particular step in
assembly could be affected by regulators and coactivators. In the holoenzyme model,
these contacts could affect a rate-limiting step in initiation. Isolated RNAP II
holoenzymes have been shown to respond to some activators including Gal4-VP16 and
GCN4 (Kim, Y.-J. et al., 1994). Serum response factor regulates transcription through
the RAP74 subunit of TFIIF (Zhu and Prewes, 1994; Joliot et al., 1995). Since RAP74
functions in initiation, promoter escape, and elongation, serum response factor may
regulate each of these processes. The transcriptional activation domain of VP16 interacts
with TFIIH (Xiao et al., 1994), and this contact may stimulate the initiation helicase to
promote open complex formation (Jiang et al., 1994). Open complex formation is an
important target for bacterial activators (reviewed by Gralla, 1990).

Overview

30
Understanding the mechanism and regulation of RN AP H requires description of

the functions of factors in transitions between stages of the transcription cycle. Our
laboratory has focused on functions of TFIIF. Cloning cDN As encoding human RAP30
and RAP74 has facilitated these studies (Sopta et al., 1989; Finkelstein et al., 1992; Wang
et al., 1993, 1994). An extract depleted of these factors has been used to assay TFIIF
function in vitro (Burton et al., 1986, 1988; Finkelstein et al., 1992; Chang et al., 1993:
Wang and Burton, 1995; Chang and Burton, 1995). Bacterial production of RAP30 and
RAP74 proteins has provided active TFIIF for reconstitution studies. Deletion mutants of
TFIIF subunits have been constructed to relate functional domains to protein sequence.

As described in chapter 11, sarkosyl challenge and pulse-chase assays were used to
distinguish the requirements of TFIIF subunits for initiation and elongation. RAP30 was
essential to establish a sarkosyl-resistant complex, indicating that RAP30 is required for
initiation. RAP74 was not required for creation of a sarkosyl-resistant complex, but
surprisingly, strongly stimulated transcription when added after sarkosyl. The same
result was observed with a pulse-chase protocol in which accurately initiated RNA is
labelled during a short pulse, followed by chase with excess unlabelled nucleoside
triphosphates. RAP30 was required to label the transcript during the pulse, but RAP74
was not. RAP74, however, was required to observe the runoff RNA. Therefore, RAP30
and RAP74 appear to have separable functions in this extract system. The RAP30
subunit supported all initiation functions of TFIIF. The RAP74 subunit was essential for
early elongation of RNAP II transcription.

To follow up these observations, transcription was initiated on an immobilized
DNA template so that the shortest stable transcripts could be identified. Short nascent
RNA complexes were found to be initiated accurately from the Adenovirus major late
promoter and paused 11-20 nucleotides from the initiating base. Synthesis of short RNAs
requires ATP hydrolysis, and both RAP30 and RAP74. These complexes are paused and

not arrested because they can be quantitatively chased to the runoff position. In testing

31
the stability of these complexes in the presence of RAP74 and RAP74 mutants, these

complexes were very stable if initiated in the presence of RAP74. Deletion of RAP74 C-
terminal sequences decreased the stability of these complexes. The ability of RAP74 to
stabilize short transcripts may partially explain the requirement for this factor in promoter
escape.

The final chapter of this dissertation describes preliminary experiments with a
protein isolated from yeast that may be a component of an alternate form of TFIIF. The
amino acid sequence of Cdc73p has been compared to that of human RAP30 and
bacterial sigma factors by Shi Min Fang of our laboratory. This sequence comparison
allowed us to predict a region of Cdc7 3p that might be involved in RNAP H binding.
Cdc73p was found to bind directly and stoichiometrically to polymerase, and a mutant
with a 15 amino acid deletion within the predicted polymerase binding site was severely

inhibited for RNAP H binding.

32
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CHAPTER II

RAP30/74 (TFIIF) IS REQUIRED
FOR PROMOTER ESCAPE BY RNA POLYMERASE II

44

45
ABSTRACT

RAP30 and RAP74 are subunits of the transcription factor called variously
RAP30/74, TFHF, By and PC. This factor is required for accurate transcription by RNA
polymerase H, in addition to other basal transcription factors. Using recombinant human
RAP30 and RAP74, the functions of these subunits have been tested separately during the
initiation and elongation phases of transcription. RAP30 is required to form a sarkosyl-
resistant complex at 0.25% sarkosyl, so RAP30 is required for initiation. RAP74,
however, stimulates transcription when added after sarkosyl, indicating that RAP74 is
dispensible for initiation. The same result is obtained using a pulse-chase protocol in
which accurately initiated RNA is labeled during a short pulse, followed by a chase with
excess unlabeled nucleoside triphosphates. RAP30 is required in order to label the
transcript during the pulse, but RAP74 is not. RAP74 must be added during the chase,
however, in order to obtain a short run-off transcript. The following conclusions can be
drawn from these experiments: 1) RAP30 is an initiation factor; 2) RAP74 is not required
for ATP hydrolysis in initiation, which precedes phosphodiester bond formation; 3)
RAP74 is not required for template strand separation; 4) RAP74 is not required to initiate

phosphodiester bond formation; and 5) RAP74 is required for very early elongation.

46
INTRODUCTION

To accurately express genetic information, RNA polymerases select promoter
sequences with precision. Because of this requirement, polymerases interact with
initiation factors that direct them into a tight association with the promoter. RNA
polymerases must then make a transition from being a sequence-specific DNA binding
protein, with high selectivity for promoter sites, to an elongation enzyme that has little
ability to discriminate between different sequences. The transition between a pre-
initiation complex, an initiated complex and an elongation complex involves multiple
steps, including: 1) template binding in the presence of initiation factors; 2) separation of
DNA template strands; 3) formation of phosphodiester bonds; 4) alterations in
polymerase conformation; 5) dissociation of polymerase from sequence-specific DNA
binding initiation factors; and 6) stabilization of an elongation conformation of RNA
polymerase by elongation factors (reviewed in 1, 2).

In the Gram-negative eubacterium E. cali, mechanistic details of the transition
from an initiated to an elongation complex have been elucidated. E. cali RNA
polymerase initiates transcription in the presence of a sigma initiation factor. The
primary sigma factor in E. cali is called 0'70; this initiation factor has an RNA
polymerase binding domain and sequence-specific DNA-binding domains that separately
recognize the -10 (TATAAT) and -35 (TI‘GACA) regions of promoters. 0’70 first binds
to RNA polymerase before associating with the promoter. Binding of sigma releases
RNA polymerase from non-specific sites on DNA, and binding to polymerase alters 0’70
conformation to expose DNA-bindin g domains (1, 3). In the presence of its sigma factor,
RNA polymerase binds tightly to promoter DNA, first to form the closed complex, which
is later opened by separation of the template DNA strands. At many promoters RNA
polymerase then synthesizes numerous short transcripts before entering a productive

elongation mode. This process is termed abortive initiation: a process that represents

47
repeated re-initiation by RNA polymerase without escape from the promoter. A change

in RNA polymerase conformation is required that causes 070 release, allowing
polymerase escape (2, 4). This elongation conformation of RNA polymerase is stabilized
by binding elongation factors such as N usA protein.

Despite evolutionary conservation of RNA polymerases and domains of some
initiation factors, the initiation mechanism and the transition from an initiated complex to
an elongation complex is less well understood in mammalian transcription. Human RNA
polymerase 11 must interact with a number of general factors, including TFHD, TFHB,
TFHF (RAP30/74), TFIIE, TFIIH and TFIIJ, in order to accurately initiate transcription
from a promoter (reviewed in 5). T FHD binds to the TATAAA region of the promoter
followed by association of TFHB (DB complex). RNA polymerase H binds first to the
RAP30 subunit of TFIIF (6). This binding suppresses non-specific DNA binding by
RNA polymerase II (7, 8) and is required for stable association of RNA polymerase H
with the promoter (6, 9). In keeping with its O7O-Iike functions, human RAP30 is
homologous to 0'70 within its RNA polymerase-binding domain (10, 11). DNA
recognition roles of 070 appear to be a function of TFHD and TFHB and may be reflected
in a weak sequence similarity between the TATAAA binding domain of TFIH) and the
TATAAT binding domain of O70 (12). After formation of the DBPolF complex
(complex of template DNA, TFIH), TFIIB, RNA polymerase II and TFIIF), TFIIE,
TFIIH and TFHJ can be incorporated, forming the completely assembled pre-initiation
complex (5). ATP hydrolysis is required by RNA polymerase H prior to template strand
separation (13) and phosphodiester bond formation (14). After synthesis of the fast
phosphodiester bonds, RNA polymerase must escape the strong protein-protein and
protein-DN A contacts holding it to the promoter. The mechanism by which this occurs is
not known. Factors that remain bound at the promoter as polymerase exits, factors that
dissociate from polymerase and the promoter, and factors that remain associated with

polymerase during elongation have not been clearly determined.

48
One feature of the transition from a pre-initiation complex to an elongation

complex has been proposed to involve covalent modification of RNA polymerase H
(reviewed in 5, 15). At the COOH-terminal end of the largest subunit of RNA
polymerase H (homolog of the 8’ subunit of E. cali RNA polymerase) is an unusual
carboxy terminal domain (CTD) which consists of 52 repeats of the consensus
heptapeptide sequence YSPTSPS. This domain is multiply phosphorylated at the two SP
sequences on serine. The dephosphorylated form of RNA polymerase H is referred to as
the Ha form, and the multiply phosphorylated form as the Ho form. RNA polymerase H
binds to the DB complex in the Ila form, at least in part because the dephosphorylated
CTD binds to the TBP (T ATA binding protein) subunit of TFIH) (16). Within the pre-
initiation complex, the CTD becomes phosphorylated by a stably associated CTD kinase
(17). The phosphorylated CTD does not bind TBP, facilitating release of RNA
polymerase H from the promoter ( 16).

Phosphorylation of the CTD does not account for the ATP requirement in
initiation because: 1) accurate transcription using RNA polymerase H lacking a CTD
requires ATP hydrolysis; 2) GTP will support CTD phosphorylation but not initiation;
and 3) the ATP concentration requirement for initiation is significantly higher than that
for conversion of 11a polymerase to the 110 form (16). The Ho form is the normal
elongation form of RNA polymerase II.

In this report, we identify another feature of the transition from the pre-initiation
complex to the initiated complex and elongation complex. This transition involves the
activity of the heteromeric transcription factor RAP30/74. We demonstrate that the
RAP30 subunit will support all of the initiation functions of this factor. The RAP74
subunit is, however, dispensible for initiation functions and instead is required for

promoter escape by RNA polymerase 11.

49
MATERIALS AND METHODS

DNA templates for transcription assays

Plasmid pSmaF contains the Smal-F fragment of Adenovirus-2 subcloned into the
Smal site of pBR313 (18). When this plasmid is digested with Smal, the accurately
initiated transcript from the Adenovirus major late promoter (AdMLP) is 536 nucleotides
in length. pSmaF digested with Smal was used as the template for the experiments
shown in Figures 1-4.

Plasmid pML was constructed by subclonin g the AdMLP as an XhoI to HindHI
fragment (coordinates -256 to +196 relative to the AdMLP cap site) between the XhoI
and HindIH sites of the vector pBluescript 11 KS (+) (Stratagene). The resulting plasmid
has a single Smal site located at +217 relative to the AdMLP cap site. This template
digested with Smal was used for the experiments shown in Figures 5 and 6.

Extract systems for in vitro transcription

An extract derived from HeLa cell nuclei was prepared as described in Shapiro et
al. (19). A RAP30/74 depleted extract was prepared as previously described (20, 21).
Affinity-purified anti-RAP30 antibodies were mixed with the extract and then the
solution was passed through a protein A-Sepharose column to remove RAP30/74 and
antibodies from the solution. This antibody depletion was repeated a second time to
remove any residual RAP30/'74 from solution. This solution was then passed through
two protein A-Sepharose columns to remove any remaining RAP30/74 and anti-RAP30
antibodies from the extract. The extract was then concentrated by centrifugation using a
Centricon-IO microconcentrator (Amicon). The resulting extract is functionally depleted
of both RAP30 and RAP74 (Figures 1-6). The extent of RAP30 depletion of the
RAP30/74 depleted extract has previously been documented (20).

Recombinant RAP30 and RAP74

50
Methods for preparation of recombinant human RAP30 and RAP74 have been

published elsewhere (22). RAP30 used in these experiments was purified to near
homogeneity. RAP74 used in these experiments is a mixture of full length RAP74 and
RAP74 fragments. RAP74 concentrations were estimated by Coomassie blue staining of
gels and reflect the amount of the intact protein.
In vitro transcription assays

The methods for in vitro transcription have been published previously (23).
Reactions were done at 30 0C. AdMLP DNA was at 60 ug/ml in all reactions. Pre-
incubations were done in 20 1.11. For Sarkosyl block and pulse-chase reactions, initiating
nucleoside triphosphates were added in 2 111. For sarkosyl block procedures, 2 111 of a 4
% sarkosyl solution was added to reactions, to a concentration of 0.33%. Elongating
nucleoside triphosphates were added in 5 111 for all procedures. After addition of
elongating nucleoside triphosphates, sarkosyl was diluted to 0.28 %. Detailed procedures
for individual experiments are given in the figures and figure legends. Ot-amanitin was
added to reactions to a final concentration of 1 rig/ml where indicated. Purification of
RNA for electrophoresis has been described in detail previously (23). Transcripts were
resolved in 6% polyacrylamide gels containing 50% (w/v) urea, and visualized by
autoradiography. Quantitation of accurate transcription was done using a Molecular

Dynamics Phosphorimager as described in (22).

5 1
RESULTS

RAP30 and RAP74 are required for accurate transcription from the adenovirus

major late promoter

A RAP30/74-depleted extract (RAP30/74-DE) was prepared by immunodepletion
using anti-RAP30 antibodies (20, 21). This extract was not active for accurate
transcription unless it was supplemented with RAP30 and RAP74 (Figure 1). The
template for runoff transcription is Adenovirus major late promoter (AdMLP) DNA
digested with restriction endonuclease Smal. The runoff transcript from the AdMLP is
536 nucleotides. In this report, RAP30 and RAP74 used in reconstitution assays were
produced in E. cali using human cDN As subcloned into bacteriophage T7 expression
vectors (22). When RAP30 was omitted from the reconstituted system, no 536 transcript
was observed (lanes 5, 7, 9 and 11). Similarly, omission of RAP74 fiom the reconstituted
system resulted in loss of the 536 runoff transcript (lanes 5 and 6). Apparently both
subunits of this factor are essential for initiation and/or elongation of this short runoff
transcript.

The level of transcription observed in the fully reconstituted system is similar to
that observed for the intact transcription system before immunodepletion (compare lane
12 with lanes 1-4). Addition of RAP30 and RAP74 to the intact system did not stimulate
transcription substantially (lanes 2-4), indicating that these factors are not limiting in the
untreated extract.

To test whether the requirement for RAP30 and RAP74 was during the initiation
or elongation phases of transcription, RAP30 and RAP74 were added back to a

RAP30/74 depleted extract in sarkosyl block and pulse-chase protocols (see below).

52

Figure 1. Both RAP30 and RAP74 are required for accurate transcription from the
AdMLP. Additions to each reaction are indicated at the top of the figure. NE) Extract
derived from HeLa cell nuclei; RAP30/74 DE) RAP30/74 depleted extract. 100 ng
recombinant RAP30 was added where indicated. Amounts of RAP74 added into various
reactions are in ng. The template for runoff transcription was pSmaF DNA digested with
Smal. The accurately intiated runoff transcript from the AdMLP is 536 nucleotides. The

reaction protocol is shown at the bottom Of the figure.

53

 

m++++
mull ++++++++
m + + + + + +
6565

9" r: l. ‘

bee-r- W nae-r-

Figure 1

RAP

sarkl

abs
sari

the

nut

Ilut

(la

54
RAP30 and RAP74 are required for accurate transcription in the presence of

sarkosyl

At concentrations higher than 0.25 %, the ionic detergent sarkosyl completely
eliminates re-initiation by RNA polymerase II (24). The requirements for forming a
sarkosyl-resistant complex are: 1) hydrolysis of the M phosphate bond of ATP; and 2)
formation of the first few phosphodiester bonds of the RNA chain (24, 25). Since
initiation must precede formation of the sarkosyl-resistant complex, this detergent has
been used to discriminate between initiation and elongation functions: a factor that is
required for transcription prior to addition of sarkosyl is an initiation factor; a factor that
is required for transcription but can function when added after sarkosyl is an elongation
factor, since no new initiation can occur after addition of the detergent.

Because the requirements for transcription elongation might be different in the
absence or presence of sarkosyl, we tested the requirement for RAP30 and RAP74 in a
sarkosyl block protocol (Figure 2). When the transcript is elongated in the presence of
the detergent, both RAP30 and RAP74 must be added to restore accurate transcriptional
activity to a RAP30/74 depleted extract. The reaction scheme is shown at the bottom of
the figure. The fully reconstituted system is shown in lanes 1 and 2. In the presence of
high sarkosyl, RNA polymerase II pauses at position +186 from the adenovirus major late
promoter (AdMLP) (24). The runoff transcript, which is not observed, would be 536
nucleotides in length. In the presence of 1 rig/ml or-amanitin, production of the 186
nucleotide transcript is abolished, showing that it was synthesized by RNA polymerase H
(lane 3). In the absence of RAP30 and RAP74 no transcript was produced (lanes 4 and
5). Neither RAP30 (lane 6) nor RAP74 (lane 7) alone restored accurate transcriptional
activity to the depleted extract. Therefore, both RAP30 and RAP74 are required for

transcription in the presence of sarkosyl.

55

Figure 2. Both RAP30 and RAP74 are required for accurate transcription in the
presence of sarkosyl. HK + glc) 10 uU hexokinase and 50 uM D-glucose; Amanitin) 1
rig/ml a-amanitin. The 186 nucleotide paused, accurately initiated transcript from the
AdMLP is indicated. RAP30/74 depleted extract was incubated with AdMLP template.
RAP30 (100 ng), RAP74 (200 ng) and other additions were made at t = -60 min.
Initiating nucleotides: 100 11M ATP, CTP and UTP (t = -1 min). Sarkosyl was added to
0.33%. Elongating nucleotides: 600 uM ATP, CTP, UTP and 25 uM a32P-Grp.

Elongation was allowed to proceed for 40 min.

56

RAP30/74m +++++++

 
     
   

RAP30+++ +
W4+++ +
HK+GIc ++ +
A-dth

Ill

3456

t=0 min
Sarkosyl

I=-I min [:44 [Illa
M-MLP DNA A,C,U C,U,‘G
RAT DE STIOP

Figure 2

11 var
'6 ShC
AIM
fillet

57
Since initiation of transcription requires ATP hydrolysis, hexokinase and D-

glucose were added to some of the reactions (lanes 1, 3, 4, 6 and 7) to deplete ATP and
confine initiation events to a 1 min window prior to the addition of sarkosyl. This
treatment renders the extract completely dependent on the addition of [5—7 hydrolyzable
ATP for initiation (data not shown). Without these additions, low levels of contaminating
ATP and other nucleoside triphosphates might allow initiation during the pre-incubation.
In the experiments shown in Figures 3 - 6, hexokinase and D-glucose (50 11M) have been

added to the extract prior to the pre-incubation.

Requirement for RAP30/74 function before sarkosyl addition

In Figure 3A, recombinant RAP30/7 4 was added to a RAP30/74 depleted extract
at various times before or after addition of sarkosyl (open squares). The reaction protocol
is shown at the bottom of the figure. The MP30/74 depleted extract was incubated with
AdMLP template for 1 hr. At time = -1 min, 100 uM ATP, CTP and UTP were added to
allow ATP hydrolysis and formation of the first 9 phosphodiester bonds from the Ad-
MLP (pppACUCUCUUCCG). Sarkosyl is added at t = 0 min. At t = +1 min, 600 11M
ATP, CTP and UTP are added along with 25 uM a-3ZP GTP to allow elongation and
radiolabeling of the transcript. RAP30/7 4 was added at the indicated times before or after
addition of sarkosyl. The +186 AdMLP transcript was quantitated using a B-radiation
scanner, and accurate transcription is reported in arbitrary units.

Strong stimulation of accurate transcription was only observed when RAP30/74
was added before addition of sarkosyl, so RAP30/74 is an initiation factor. Based on this
analysis, either RAP30, RAP74 or the RAP30/74 complex is required for initiation. The
kinetics of stimulation of transcription by recombinant RAP30/74 is very similar to that
previously reported for human RAP30/7 4 (20). The recombinant proteins are efficiently

incorporated into the pre-initiation complex in a rapid step, late in the assembly pathway.

Figur
PAP}
(300 l
38, l
was 2
R1?
RAF
20, 3

Sarkl
25 p
Slap;

for b
the c‘

liar.

58

Figure 3. Kinetics of RAP30 and RAP74 function in transcription. Panel A)
RAP30/74 and RAP30 are initiation factors. (open squares) RAP30 (100 ng) and RAP74
(200 ng) were added at the indicated times (t = -60, -40, -20, -10, -5, -2, -1, 0, 1, 2, 5, 10,
38, 40 min). (closed circles) RAP74 (200 ng) was added at -60 min; RAP30 (100 ng)
was added at the indicated times (t = - 10, -5, -2, -1, 0, 1, 2, 5, 10, 40 min). Panel B)
RAP74 is an elongation factor. (open circles) RAP30 (50 ng) was added at t = -60 min;
RAP74 was added at the indicated times (t = -60, -40, -20, -10, -5, -2, -1, 0, 1, 2, 5, 10,
20, 38, 40 min). A RAP30/74 depleted extract was incubated with AdMLP DNA for 60
min. At t = -1 min, ATP, CTP and UTP were added to 100 11M to initiate transcription.
Sarkosyl was added to 0.33 % at t = 0 min. At t = +1 min, 600 11M ATP, Cl'P, UTP and
25 uM a32P-GTP were added to allow elongation of transcription. Reactions were
stopped at t = +40 min. The data presented in B is the combination of two experiments
scaled together by making the -10 min time points equivalent in transcriptional activity
for both experiments. Apparent scatter in the data reflects a slightly different kinetics in
the dip of transcriptional activity seen near the time of sarkosyl addition. The shapes of
curves for both experiments were very similar. The paused 186 nucleotide transcript was

quantitated as described in Materials and Methods.

ACCURATE TRANSCRIPTION

(ARBITRARY UNITS)

59

 

 

 

a RAP30/74
10° ‘ A. e RAP30
80 -
60 it
40 -
I.
20 -

 

 

 

 

 

0 v I V T ' I ' I f
-60 ~40 ~20 0 20 40
TIME (MIN)
t=0 min
Sarkosyl
tz-l min t=+l min

Ad-MLP DNA A,C,U ,C,U,*G
RAP30/l: DE STIOP

Figure 3

RAPE

iir'ltos
prior ‘
trio ll
' 'rrcti

trait

Rll’

PAP

but I

don;

mile

Obge

"as

thn‘

POin

ACCC

60
In Figure 3A, we show that RAP30 is an initiation factor (filled circles).

RAP30/74 depleted extract, supplemented with RAP74, was mixed with template DNA at

= -60 min. RAP30 was added to the reaction at various times before or after addition of
sarkosyl. Accurate transcription was strongly stimulated only when RAP30 was added
prior to sarkosyl, so RAP30 is an initiation factor. The kinetics of RAP30 assimilation
into the pre-initiation complex is indistinguishable from assembly of RAP30/74. The
kinetics of assembly of recombinant RAP30/74 and RAP30 is indistinguishable from that
previously published for human RAP30/7 4 (20).

RAP74 is dispensible for initiation but required for elongation

A more startling observation was made in the experiment shown in Figure 3B.
RAP74 was required for accurate transcription, as previously shown in Figures 1 and 2,
but RAP74 strongly stimulated transcription whether it was added before or after
sarkosyl. By this analysis, RAP74 is not required for initiation; rather, RAP74 is an
elongation factor.

In this experiment, RAP30fl4 depleted extract, supplemented with RAP30, was
mixed with template DNA and pre-incubated for 1 hr. RAP74 was added at various
times before or after addition of sarkosyl (open circles). When RAP74 was added at the
time of sarkosyl addition or after sarkosyl addition, significant accurate transcription was
observed. If RAP74 was never added (t = +40 min), no transcription from the AdMLP
was observed. Even when RAP74 was added at t = +38 min, leaving only 2 min for
elongation, 20 % of the +186 transcript was observed relative to the t = -10 min time
point. Apparently, RAP74 can function in early elongation even in the presence of
sarkosyl.

Most surprisingly, however, RAP74 is dispensible for initiation functions.

According to this analysis, RAP74 is an elongation factor that is absolutely required to

Wht
Com
of a
Flor
the l
the
ill

is pr

61
synthesize a short (186 nucleotide) transcript. RAP74 is, therefore, not required for: 1)

pre-initiation complex formation; 2) ATP hydrolysis in initation; 3) template strand
separation; or 4) first bond formation. It is only required after these processes are
completed, and the sarkosyl-resistant complex is formed. RAP74 is, however, required to
synthesize a short runoff transcript from the AdMLP. RAP74 appears to be required for
promoter escape by RNA polymerase II.

The dip in transcriptional activity seen when RAP74 is added along with sarkosyl
(t = 0 min) or one minute after sarkosyl (t = +1 min) has been reproducible in replicate
experiments. Apparently, RAP74 activity is most sensitive to sarkosyl at the time of
early transcript elongation. When added before initiation (t = -20 to t = -5 min), RAP74
may be protected from sarkosyl inhibition by incorporation into the pre-initiation
complex (6). Also, RAP74 may have completed its function in promoter escape before
addition of detergent. More puzzling is the increase in transcriptional activity when
RAP74 is added just after sarkosyl (t = +2 to +10 min). Perhaps sarkosyl dissociates a
factor that destabilizes the ternary complex (ie. TFIIS, see Discussion). Also, the
effective concentration of sarkosyl may decrease with time after addition, as detergent
binds proteins in the extract. Detergent concentration was slightly decreased by addition
of nucleoside triphosphates at t = +1 min. The decrease in accurate transcription seen
when RAP74 was added at t = +5 to +38 min probably reflects both instability of ternary
complexes and shorter elongation times. Apparent scatter in data points around the time
of addition of sarkosyl is due to combination of data from two separate experiments to
produce the graph reported in Figure 3B. Scatter represents slightly different kinetics of
the dip in transcriptional activity around the time of sarkosyl addition. In any case, all of
the data collected in replicates of this experiment supports our major conclusion, that
RAP74 is an elongation factor (see also Figure 4).

In Figure 4, the surprising result that RAP74 is dispensible for initiation functions
is presented as gel data. RAP30/74 depleted extract, supplemented with RAP30, was

62

Figure 4. RAP74 is an elongation factor. The reaction protocol was the same as shown
in Figure 3B. RAP30 (100 ng) was added at t = -60 min; RAP74 (200 ng) was added at t

= -10 min, +10 min or omitted altogether, as indicated.

63

 

 

 

 

 

 

 

 

 

 

 

331-

 

 

 

 

 

Figure 4

mixed V
(has 1
min). I
tarsal
after ad

RS 0b:

Conflr

in Fl g1
instill
abscn

tlper

PIC-i:

Cha

64
mixed with AdMLP template DNA. RAP74 was added to the extract at t = ~10 min

(lanes 1 and 2) or at t = +10 min (lanes 3 and 4), relative to addition of sarkosyl (t = 0
min). In lanes 5-8 RAP74 was never added to the reaction (t = +40 min). Accurate
transcription was strongly stimulated when RAP74 was added to the reaction before or
after addition of sarkosyl. In the absence of RAP74 addition, no accurate transcription

was observed. Clearly, RAP74 is an elongation factor.

Confirmation of the sarkosyl results using a pulse-chase protocol

One possibility that we wished to investigate was whether the observations made
in Figures 3 and 4 were somehow limited to transcription reactions done in sarkosyl. For
instance, sarkosyl might stabilize a short ternary complex that would be unstable in the
absence of detergent. RAP74, for instance, might appear to be an initiation factor in an
experiment that did not include sarkosyl.

In Figure 5, RAP30 is shown to be an initiation factor in a protocol that does not
include sarkosyl. The RAP30/74 depleted extract was mixed with template DNA and
pre-incubated for 60 min. RAP74 was added to the reaction at t = -5 min. RAP30 was
added to the reaction at t = -5 min, t = +5 min or omitted altogether, as indicated in the
figure. Initiating nucleoside triphosphates ATP, UTP (100 uM each) and a-32P CTP
(625 nM) were added at t = -1 min. At t = 0 min elongating nucleoside triphosphates
ATP, UTP, CTP and GTP (1 mM each) were added. The >1000 fold excess of un-
labeled CI'P added during the chase eliminated radiolabeling of transcripts initiated after t
= 0 min. The accurately initiated runoff transcript from the AdMLP in this assay is 217
nucleotides.

Accurate transcription was strongly stimulated when RAP30 was added at t = -5
min, before the pulse and chase (lanes 3 and 4). Because of high backgrounds in pulse-

chase experiments, critical points were done in duplicate. This 217 nucleotide transcript

Figur

65

Figure 5. Pulse-chase protocol: RAP30 is an initiation factor. RAP74 (200 ng) was
added at t = -5 min, as indicated; RAP30 (100 ng) was added at t = - 5 min or at t = +5
min, as indicated. A RAP30/74 depleted extract was mixed with AdMLP DNA at t = ~60
min. Pulse nucleoside triphosphates: 100 uM ATP, UTP and <1 11M a32P-CTP (10
uCi). Chase: 1 mM ATP, CTP, UTP and GTP. The accurately initiated runoff transcript

is 217 nucleotides.

      

RAP30/74 DE

RAP30
(TIME)
Chase before Pulse

Amanitin

A.U,C‘(-I min) Cline (0 Inln)
AdMLP DNA

 

+10
RAP30m/74 DE A +15
L__.l
-60

-s
RAP74 *5

Figure 5

is a

and
sho
new
did

prlo
trans
madl

lnitia

luclt

pulse-

the in
Wk
R1177
+5 mi
before
MP7

liters

67

is abolished in the presence of 1 rig/ml a-amanitin (lane 1). When the order of the pulse
and chase was reversed (lane 2), this also abolished detection of the accurate transcript,
showing that sufficient cold CTP was added during the chase to eliminate labeling of
newly initiated transcripts. Addition of RAP30 at t = +5 min, after the pulse and chase,
did not strongly stimulate transcription (lanes 7 and 8). Since RAP30 must be added
prior to addition of chase nucleotides, in order to incorporate 32P-CMP into the
transcript, RAP30 must be an initiation factor. These results confirm the observations
made in Figure 3A using a sarkosyl block protocol: RAP30 is required for accurate
initiation of transcription.

Omission of RAP74 from the reaction (lanes 9 and 10) also abolished the 217
nucleotide transcript, showing that RAP74 is required for accurate transcription in the
pulse-chase protocol.

In the experiment shown in Figure 6, RAP74 is tested for its requirement during
the initiation and elongation phases of transcription. The RAP30/'74 depleted extract,
supplemented with RAP30, was mixed with template DNA and pre-incubated for 60 min.
RAP74 was added to the reaction at either t = -5 min (before the pulse and chase) or at t =
+5 min (after the chase). RAP74 strongly stimulated transcription whether it was added
before the pulse (lanes 3 and 4) or after the chase (lanes 7 and 8). From this analysis,
RAP74 is an elongation factor. RAP74 is required for accurate transcription, but only
after synthesis of an accurately initiated RNA has occured.

As expected, omission of RAP74 from the reaction abolished accurate
transcription (lanes 9 and 10). Omission of RAP30 from the reaction abolished accurate
transcription (lanes 11 and 12). Reversing the order of the pulse and chase (lanes 2 and
6) abolished visualization of the transcript, and the transcript was completely abolished

by addition of l rig/ml a-amanitin.

Figu
aide

isln

68

Figure 6. Pulse-chase protocol: RAP74 is an elongation factor. RAP30 (100 ng) was
added at t = -60 min, as indicated. RAP74 (200 ng) was added at t = -5 rrrin or at +5 min,

as indicated. All other reaction parameters were as described in Figure 5.

umwri++++++++++++

+
+
+
+
+
+
+
+
+
4..

(mm -5 -5 -5 -5 +5 +5 +5 +5 -5 -5

 

 

69

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

141 o
111
110
0 00000000.
67-. -
. "f r- :
,’, 3 i I ‘L i I
M123456fi9101112
AdMLP DNA A,U,C‘(~1-ln) Clue (0 in)
RAP30/7408 +1.
I / J +15
l T___l
"‘ -5 +5

 

Figur 6

DIS(

lull:

milj

and]
hktl

don;

mda
P3PC

llOl'll

Poly

ahhc
less
Cour
Phos
Irans

70
DISCUSSION

We have separately analyzed the functions of RAP30 and RAP74 during the
initation and early elongation phases of transcription by RNA polymerase H. We find
that RAP30 is an essential initiation factor, but that RAP74 is dispensible for initiation
functions and is only required for very early elongation of the transcript. Since RAP30
and RAP74 are tightly associated in a heteromeric complex (26-28), these factors most
likely enter the pre-initiation complex together, bound to RNA polymerase 11.
Surprisingly, RAP30 and RAP74 have separate functions in accurate initiation and early
elongation of RNA chains.

In Figure 7 we present a model for the function of RAP30 and RAP74 during
intiation and elongation of transcription. This model is based on data presented in this
paper and also the work of many others (reviewed in 5). The RAP30/74 complex
normally enters the pro-initiation complex bound to RNA polymerase H. RAP30/74
binds to RNA polymerase H primarily through the RAP30 subunit (8, 11), and the
RAP74 subunit is not required to recruit RNA polymerase H into the pre-initiation
complex (6). Our observation that RAP30 is required for initiation of transcription
(Figures 3A and 5) is consistent with evidence that RAP30 helps to bring polymerase to
the promoter (6, 9). ATP hydrolysis is required for accurate initiation by RNA
polymerase H (14, 25, 29, 30), and hydrolysis precedes formation of phOSphodiester
bonds (14). Sensitivity of transcription complexes to KMnO4 indicates that ATP
hydrolysis is required to separate template strands to form the open complex (13),
although studies with o—phenanthroline copper to detect open complex formation seem
less consistent with this conclusion (31). In any event, resistance of transcription
complexes to sarkosyl requires both ATP hydrolysis and formation of the first few
phosphodiester bonds (24, 25). In this paper we show that RAP74 is not required for
transcription until after addition of sarkosyl (Figures 3B and 4), showing that RAP74 is

View

71

Figure 7. A model for RAP30/74 function in initiation and early elongation of
transcription. RAP30/7 4 normally enters the pre-initiation complex bound to RNA
polymerase H, but the RAP74 subunit is dispensible in this process. The RAP30 subunit
is required to bring RNA polymerase 11 into the pre-initiation complex. RAP74 is not
required for transcription until after: 1) ATP B-y bond hydrolysis in initiation; 2) open
complex formation; and 3) formation of the first few phosphodiester bonds. RAP74 is
required for RNA polymerase H to escape from the promoter. This model represents our

view based on the work of many investigators (see the text for details).

72

 

 

 

 

 

TFHD+TFHA TFHB
Promoter DNA > DA complex \ >DAB complex
RAP30-RNAPH
complex
RAP74 normally
DABPol RAP30 complex enters here
E
FIIH
TFIIJ

DABPOIRAP EHJ complex
(closed complex)
ATP

ADP+Pi

DABPolRAP30EI-IJ complex
(open complex)

ATP,CTP,UTP

Initiated complex
(Sarkosyl resistant)

 

 

RAP74 is not required before this step

 

 

ATP,CTP,UTP,GTP

ELONGATION
COMPETENT COMPLEX

Figure 7

onis
posil
ape

piles

nutl
PIES
any
to I]
sho
abs
css:
R1

tOI

the
of

no

73
only required after ATP hydrolysis, DNA strand separation and phosphodiester bond

formation have occured. Using a pulse-chase protocol (Figure 6), we show that RAP74 is
only required after formation of a short RNA chain (fewer than 11 nucleotides by
omission of GTP) labeled by incorporation of 32P-CMP (C can be incorporated into
positions 2, 4, 6, 9 and 10 of the chain in the absence of GTP). The pulse-label
experiment shows that RAP74 is dispensible for template strand separation and
phosphodiester bond formation. After these steps in initiation have occured, RAP7 4 must
be added to the reaction for RNA polymerase H to synthesize a short runoff (217 or 536
nucleotides) or paused (186 nucleotide) transcript. Inspection of transcripts formed in the
presence of all 4 nucleoside triphosphates but in the absence of RAP74 does not reveal
any short paused transcripts (see Figure 1). Attempts to observe short RNAs very close
to the promoter on 20 % polyacrylamide gels have so far not been successful (data not
shown). Apparently, RNA polymerase II is stalled very close to the promoter in the
absence of RAP74. Our conclusion from these observations is that RAP74 has an
essential function in promoter escape by RNA polymerase H. We think it likely that the
RAP30 subunit of RAP30/7 4 participates in promoter escape, at least to help bind RAP74
to polymerase.

Although the RAP30/74-depleted extract is functionally depleted of RAP30 and
RAP74 (Figures 1-6), we cannot be certain that we have removed all of the RAP74 from
the extract. Residual RAP74 might be sufficient to support initiation but not elongation
of transcription. RAP74 might, therefore, have an essential function in initiation that is
not detected in these assays, and our conclusion that RAP74 is not required for ATP
hydrolysis and intiation might be incorrect. If this alternate view were true, however, a
. higher concentration of RAP74 would be necessary for elongation than for initiation of
transcription. Either RAP74 would have to cycle off of polymerase after initiation and be
replaced by new RAP74 molecules with reduced polymerase affinity during elongation,
or a larger number of RAP74 molecules would have to be required for elongation than for

74
initiation. Such models appear to be too ornate for consideration without further

supporting evidence. In any case, such models do not contradict the major conclusion of
this work, that RAP74 has an essential function in promoter escape by RNA polymerase
H.

The RAP30 subunit of RAP30fl4 is homologous to the bacterial initiation factor
070 (10, 11). The region of sequence similarity is found within the domain of O70 that
binds to bacterial RNA polymerase (11, 32). Human RAP30 binds to E. cali RNA
polymerase and is displaced by binding of 070. O70 can also bind to calf thymus RNA
polymerase H (11). In addition to this conservation of RNA polymerase-binding sites in
RAP30 and 670, these proteins also share some functional roles in transcription. 0'70
binding to E. cali RNA polymerase suppresses non-specific DNA binding by polymerase.
RAP30 (8) and RAP30fl 4 (9) have also been shown to suppress non-specific binding to
DNA by mammalian polymerase. O70 has the additional ability to dissociate RNA
polymerase from non-specific sites on DNA to which it is bound. RAP30/74 has this
property also (8, 9); but the RAP30 subunit by itself does not (8). Both RAP30 and 0'70
are required factors for accurate initiation by their respective RNA polymerases. RAP30
does not appear to possess the sequence- specific promoter recognition properties of 070;
these properties appear to be the function of other basal and promoter-specific
transcription factors in the mammalian system.

Since the RAP30/74 complex may have an important role in release of RNA
polymerase H from non-promoter DNA sites (8, 9), RAP30/'74 may have a role in
transcription termination. A role has also been defined for RAP30/74 in bringing RNA
polymerase H to the promoter (6, 14). In this paper we show another function of
RAP30/74 in promoter escape by RNA polymerase H. Other investigators have reported
a role for RAP30fl4 in later stages of transcript elongation (33, 34) and in elongation of
RNA polymerase 11 through assembled nucleosomes (35). If RAP30/74 remains

associated with RNA polymerase 11 throughout the transcription cycle, perhaps

R11

q

fitt

elm
site
In 2

oft

site
wh
RA

cm
tlc

C0

fat

Stl

Ier

de-

75
RAP30/I4 should be considered a polymerase subunit rather than an accessory factor.

070 is considered to be an initiation factor for E. cali RNA polymerase because this
factor is catalytic in the initiation process, cycling off polymerase during elongation.
RAP30/74 is easily dissociable from RNA polymerase H at low salt concentrations (ie.
200 mM NaCl (36)), indicating that this factor may have a reversible interaction with
polymerase at some point in the transcription cycle. Perhaps RAP30/7 4 and/or other
elongation factors are dissociated from polymerase beyond splicing and polyadenylation
sites, causing polymerase to lose processivity and ultimately to terminate transcription.
In any case, RAP30/74 has been shown to have very specific functions at several stages
of the transcription cycle.

Purified RNA polymerase H can synthesize long RNA chains from non-promoter
sites when it initiates in the absence of accessory factors. In this paper, we show that
when RNA polymerase II initiates transcription from a promoter in the absence of
RAP74, the transcript is not elongated. Presumably factors present in cell extracts
regulate polymerase escape from promoters. RAP74 may function to dissociate RNA
polymerase II from strong DNA-protein and protein-protein interactions holding
polymerse at the promoter. We suggest that RAP74 participates in early elongation to
convert accurately initiated RNA polymerase H molecules from an initiated form to an
elongation competent form. RAP74 might do this by stabilizing an elongation
conformation of RNA polymerase II, and/or destabilizing interactions with other
initiation factors. Factors that regulate promoter escape may include basal initiation
factors and other proteins present in our HeLa cell extracts.

RNA polymerase H has recently been reported to have an exonuclease activity,
stimulated by the elongation factor TFIIS (37-40), that might be expected to digest short
ternary complexes, such as those formed in the absence of RAP74. Our data, however,
demonstrates that such complexes can be stable for up to 38 min in the presence of

sarkosyl (Figure 3B) and up to 5 min in the absence of sarkosyl (Figure 6). Sarkosyl

pact
Pd.
mat
hon
the

ON

C)

Si

76
likely dissociates TFHS from RNA polymerase H, and this may explain why transcription

is paused at +186 in the presence of sarkosyl (24, 41).

In Drosophila, early elongation by RNA polymerase H appears to be regulated in
part by a factor named P-TEF, which is involved in overcoming early termination of
RNA chains (42). This previous report appears to be a distinct observation from that
made in this paper, because P-TEF does not appear to be factor 5 (the RAP30/'74
homolog in Drosophila (43)), and P-TEF functions further from the promoter (42) than
the function defined for RAP74 in this paper. It may be that the human version of P-TEF
or other factors cooperate with RAP74 in regulating promoter escape.

There is mounting in viva evidence that transcription by RNA polymerase H can
be regulated at very early elongation (reviewed in 44). In Drosophila, heat shock
regulated genes have been shown to have a paused RNA polymerase very close to the
promoter prior to gene induction by temperature upshift (45). Paused polymerase is also
found just downstream of promoters for other Drosophila genes (45). The human c-myc
gene also has a stalled RNA polymerase molecule proximal to its promoter (46). Based
on the experiments in this paper, we suggest that stalled RNA polymerase H molecules
are initiated in the absence of RAP74 (or another factor that regulates promoter escape).
Promoter escape may be an important regulatory check point in control of gene
expression and should be considered a potential target for regulation by activating and
silencing transcription factors.

I-HV-l tat transactivator stimulates transcription through a tar RNA sequence, and
regulation by tat influences both initiation and elongation of RNA chains (47-50).

Regulation of elongation by tat may involve general elongation factors such as TFHS and

RAP30/74.

10

Exp
th

lttl

77

ACKNOWLEDGEMENTS

This research was supported by a grant from the National Institutes of Health
(GM 40708). Z.B. is a member of the Michigan State University Agricultural
Experiment Station. We thank Lee Kroos, Laurie Kaguni, Steven Triezenberg and Janek
Werel for critical reading of this manuscript. We thank Judith Jaehning, Donal Luse,

Jack Greenblatt and David Price for stimulating discussions and encouragement.

78
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McKnight and KR. Yamamoto, eds., Cold Spring Harbor Laboratory Press, pp. 129-178.
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McKnight and KR. Yamamoto, eds., Cold Spring Harbor Laboratory Press, pp. 179-202.
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4. Gill, S.C., Weitzel, S.E. and von Hippel, RH. (1991) J. Mol. Biol. 220, 307-324.

5. Zawel, L. and Reinberg, D. (1992) Prog. Nucleic Acids Res. Mol. Biol. 44, 67-108.

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7. Conaway, J. W. and Conaway, R. C. (1990) Science 248, 1550-1552.

8. Killeen, M. and Greenblatt, J. (1992) Molec. Cell. Biol. 12, 30-37.

9. Conaway, R. C., Garrett, P., Hanley, JP. and Conaway, J.W. (1991) Proc. Natl. Acad.
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10. Sopta, M., Burton, Z.F. and Greenblatt, J. (1989) Nature 341, 410-414.

11. McCracken, S. and Greenblatt, J. (1991) Science 253, 900-902.

12. Horikoshi, M., Wang, C.K., Fujii, H., Cromlish, J.A., Weil, RA. and Roeder, R.G.
(1989) Nature 341, 299-303.

13. Wang, W., Carey, M. and Gralla, JD. (1992) Science 255, 450-453.

14. Conaway, R. C. and Conaway, J.W. (1988). J. Biol. Chem. 263, 2962-2968.

15. Corden, J.L. (1990) Trends in Biol. Sci. 15, 383-387.

16. Layboum, RI. and Dahmus, ME. (1989) J. Biol. Chem. 263, 18880-18885.

17. Usheva, A., Maldonado, E., Goldring, A., Lu, H., Houbavi, C., Reinberg, D., and
Aloni, Y. (1992) Cell 871-881.

18. Weil, P.A., Luse, D.S., Segall, J. and Roeder, R.G. (1979) Cell 18, 469-484.

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19. Shapiro, D.J., Sharp, P.A., Wahli, W.W. and Keller, M.J. (1988) DNA 7, 47-55.

20. Burton, Z.F., Killeen, M., Sopta, M., Ortolan, LG. and Greenblatt, J. (1988) Molec.
Cell. Biol. 8, 1602~1613.

21. Finkelstein, A., Kostrub, C.F., Li, J., Chavez, D. P., Wang, B.Q., Fang, S.M.,
Greenblatt, J. and Burton, Z.F. (1992) Nature 355, 464-467.

22. Wang, B.Q., Kostrub, D.F., Finkelstein, A. and Burton, Z.F. (1993) Protein
Expression and Purification (in press).

23. Burton, Z.F., Ortolan, LG. and Greenblatt, J. (1986) EMBO J. 5, 2923-2930.

24. Hawley, D.K. and Roeder, R.G. (1985) J. Biol. Chem. 260, 8163-8172.

25. Luse, D.S., Kochel, T., Kuempel, E.D., Coppola, J. A. and Cai, H. (1987) J. Biol.
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26. Conaway, J .W. and Conaway, R. C. (1989) J. Biol. Chem. 264, 2357-2362.

27. Flores, 0., Ha, 1., and Reinberg, D. (1990) J. Biol. Chem 265, 5629-5634.

28. Katajima, S., Tanaka, Y., Kawaguchi, T., Nagaoka, T., Weissman, S. and Yasukochi,
Y. (1990) Nucleic Acids Res. 18, 4843-4849.

29. Bunick, D., Zandomeni, R., Ackerrnan, S. and Weinmann, R. (1982) Cell 29, 877 -
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30. Sawadogo, M. and Roeder, R. G. (1984) J. Biol. Chem. 259, 5321-5326.

31. Buratowski, S., Sopta, M., Greenblatt, J. and Sharp, RA. (1991) Proc. Natl. Acad.
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32. Lesley, SA. and Burgess, RR. (1989) Biochemistry 28, 7728-7734.

33. Price, D.H., Sluder, A.E. and Greenleaf, AL. (1989) Mol. Cell. Biol. 9, 1465-1475.
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Biol. 11, 1195-1206.

35. Izban, MG. and Luse, D. S. (1992) J. Biol. Chem. 267, 13647-13655.

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37. Izban, MG. and Luse, D.S. (1992) Genes and Develop. 6, 1342-1356.

38. Wang, D. and Hawley, D.K. (1993) Proc. Natl. Acad. Sci. USA 90, 843-847.

39. Reines, D., Ghanouni, R, Li, Q.-q. and Mote, J. Jr. (1992) J. Biol. Chem. 267,
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40. Kassavetis, G.A. and Geiduschek, ER (1993) Science 259, 944-945.

41. Wiest, D.K., Wang, D. and Hawley, D.K. (1992) J. Biol. Chem. 267, 7733-7744.

42. Marshall, NP. and Price, DH. (1992) Molec. and Cell. Biol. 12, 2078-2090.

43. Kephart, D. D., Price, M. P., Burton, Z.F., Finkelstein, A., Greenblatt, J. and Price,
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47. Marciniak, R.A., Calnan, B.J., Frankel, AD. and Sharp, RA. (1990) Cell 63, 791-
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48. Marciniak, RA. and Sharp, RA. (1991) EMBO J. 10, 4189-4196.

49. Kato, H., Sumimoto, H., Pognonec, P., Chen, C.-H., Rosen, CA. and Roeder, R.G.
(1992) Genes and Develop. 6, 655-666.

50. Cullen, BR. (1990) Cell 63, 655-657.

CHAPTER III

RAP74 IS REQUIRED FOR STABILITY OF NEWLY-INITIATED
TRANSCRIPTION COMPLEXES

81

ABST]

one 01
Inner
cubic
pOIyml
paused
radioal
tanscr
to tha-
transcrl
the sta
positio
Complt
tranStn'
enough
rhqune.
POSiliv

Hansen

82
ABSTRACT

The identification of structural transitions accompanying stabilization has been
one of the major concerns in the biochemical analysis of transcription. In vitro
transcription using immobilized templates and high percentage polyacrylamide gels
enable us to observe nascent RNAs 11 to 20 nucleotides in length due to stalling of RNA
polymerase II on the adenovirus major late promoter shortly after initiation. These
paused transcription complexes are mainly due to the limiting concentration of
radioactive nucleoside triphosphate; however, some intrinsic property of the basal
transcription machinery may also contribute to this stalling. This assay system enables us
to characterize the mechanism of transcription, namely the specificity of nascent
transcripts, the location of the catalytic site in the transcription complex, and formation of
the stable ternary complex. Polypeptide sequence between positions 409-517 and
positions 172-205 of RAP74 may play a critical role in stability of the transcription
complex. The most interesting observation is of a kinetic lag for the formation of stable
transcription complexes. The lag may be the effect of the short RNAs which are not long
enough to fill in the first transcript binding site of RNA polymerase H, or it may be
required for the conformational change of this enzyme to allow different interactions with
positive and negative regulatory factors. This could be a general phenomenon of

transcription and an important regulatory point in gene expression.

[\TR(

genes
sage i
Refs. '
andpr

shown

Pron

Clon
ellzl

th

83
INTRODUCTION

Both in prokaryotes and in eukaryotes, RNA polymerases (RN AP) transcribe
genes accurately. Transcription includes initiation, elongation and terminationp; each
stage in the process is a level at which gene expression can be regulated (reviewed in
Refs. 1-4). For the initiation of transcription, it includes specific binding, isomerization

and promoter escape. Schematically, the process of E. cali RNA polymerase initiation is

 

 

 

 

 

shown as.
[Speclnc blndlng J
Jo-t-RNAP RNAP- 070 L Isomerlzatlon] RNAP- g0
Promoter DN Promoter DNA __> Promoter DNA
(closed complex) (open complex)
ATP’CTP,UTP’GTP [Abortive Initiation]

 

 

Promoter Escape

Hog—l RNAP-67°

3:212:32 + arenas

NusA Abortive RNA
E .cali RNA polymerase initiates transcription in the presence of a sigma initiation
factor. The primary sigma factor in E. cali is 0'70. 0'70 first binds to RNA polymerase
before it associates with the promoter, and binding to RNA polymerase alters the
conformation of 070 to expose a DNA-binding domain (5). Also, the binding of 070
releases RNA polymerase from non-specific sites on DNA. In the presence of its 0-
factor, RNA polymerase binds tightly to promoter DNA, first to form a closed complex,
which later becomes an open complex through the separation of the template DNA
strands. At many promoters, RNA polymerase then synthesizes numerous short
transcripts, called abortive RNAs, before the RNA polymerase can become a productive
elongation enzyme. Abortively initiated RNA is released from polymerase without
enzyme dissociation from the promoter (6). Commitment to productive elongation

typically occurs after the synthesis of a 9-12-nucleotide transcript and is associated with

he rtlcasc
change in
IVA pol)
occur. It
HeLa cell
initiation

in
binding c
that occu
coll; R.‘
the modi
polymer;

pause in

Promo
35506:
00mph

18 net

Rim.

84
the release of sigma factor (7-11). Binding of RNA to the RNA binding sites (12) and a

change in conformation of RNA polymerase is required to cause 0‘70 release, allowing
RNA polymerase to escape (12). In eukaryotes, an analogous transition is believed to
Occur. It has been shown that RNA polymerase H can abortively initiate transcription in
HeLa cell nuclear extracts (13). The abortive initiation process represents repeated re-
irritiation by RNA polymerase without escape from the promoter.

In E.cali, the elongation conformation of RNA polymerase is stabilized by the
binding of elongation factors such as NusA protein (14). There are also regulatory events
that occur during elongation. One example in prokaryotes is that of phage 1 infected E.
cali; RNA polymerase pauses at position +16 on the pR' promoter of phage 2. allowing
the modification of polymerase by the phage-encoded Q protein (15). The Q-modified
POIYmerase can then leave the +16 pause site in a form that can read through subsequent
Pause and termination sites.

The essential components of messenger RNA transcription in eukaryotes are:
Promoter DNA, general transcription factors (TFHD, TFHA, TFHB, TFHF also called
RAP3O/74 [RAP is an acronym for "RNA polymerase II-associating protein"], TFIIE and
TFIH‘I), RNA polymerase H, ATP as energy source, and nucleoside triphosphates as
subStl‘ates (reviewed in Ref.1 and 2). The assembly of the pro-initiation complex on the

DNA template has been suggested to be a multistep sequential process, schematically
Shown on the next page.

In general, TFIIA stimulates the TBP binding event on TATA-containing
pronloters, then TFHB binds to form a DAB complex (16, 17). RNA polymerase H
assoCiated with RAP30/74 (TFIIF) specifically binds the promoter sequence in the DAB
complex. Although both RAP30 and RAP74 associate with polymerase in viva, RAP30
is necessary and sufficient for guiding polymerase H to the DAB complex (18, 19).

R46‘1’74 functions later during the transcription process (20, Chapter H).

Promoter

llilifill
(Sarkos

Abe

Elongat
(Stable

A
transcrip
complex
the form
lamina]
Rponed
be invol-
elongat
detachr
3 helicz
factor(:
or com
transq
exist a

open c
abom‘

Stable

85

TFHA TFHD TFHB 30/74 F)
+ RNAPH complex
Promoter DNA» DA complex» DAB compkxA-p D ABPolF complex
TFIIE
ATP,CTP Tm"
”"3" ADP+Pi An:
1m couplex DABPolFEH DABPol H complex
(Sarkosyl resistant) (open complex) (closed complex)
+
Abortive RNAs
Promoter Esacpc
(RAP74 involved)
Elongation mpetent complex» Elongation —> Termination
(Stable ternary complex)

After RNA polymerase H joins the complex, TFHB and TFIHI enter to form the
transcription closed complex (DABpolFEH complex) which is then converted to the open
complex which is characterized by the hydrolysis of ATP, the separation of DNA strands,
the formation of phosphodiester bonds, and the phosphorylation of the CTD (carboxyl
terminal domain) on the largest subunit of polymerase H (21). Recently, TFIIH has been
reported to contain a CID kinase activity and two helicase activities (22-26), which could
be involved in this event. Promoter escape occurs after initiation and before productive
elongation. It may require a conformational change of RNA polymerase II, the
detachment of RNA polymerase H from some general initiation factors, and activation of
a helicase to further unwind the DNA template. After this transition, it is not clear which
factor(s) move along with RNA polymerase H and which remain bound to the promoter
or completely dissociate from the DNA. Since commitment of RNA polymerase to the
transcription of a specific gene is a multiple step process, regulatory checkpoints could
exist at each stage, notably promoter recognition, assembly of a preinitiation complex,
open complex formation, synthesis of the first phosphodiester bond, or transition from
abortive to productive elongation. One of the important events is the formation of a

stable ternary complex of template DNA, RNA polymerase and nascent RNA. Recent

86
reports in the literature indicate that such a stable ternary complex may be a checkpoint

for transcriptional control in vivo (reviewed in 27, 28). Some RNA polymerase H
molecules have been identified as being actively engaged in transcription but also being
defective for elongation. Before heat shock on the hsp 70 promoter of Drosophila
melanogaster, an RNA polymerase H molecule is found to be initiated but stalled within
the first 29 nucleotides of the mRNA chain. After heat shock, polymerase molecules are
found distributed throughout the hsp70 gene, and the hsp70 protein is produced (29). The
human c-myc gene also appears to be regulated at the level of transcription elongation.
An attenuation site was identified for this gene at the exon I-intron I boundary, but it may
be that this site is secondary to a strong pause site located very close to the promoter (30).
A stalled RNA polymerase H molecule has been identified close to position +30 of the
human c-myc promoter by KMnO4 modification of thymidines and nuclear run-on
assays. Paused polymerase molecules at the exon I-intron I boundary may result from
elongation-defective polymerase molecules that inefficiently traverse the pause site close
to the promoter.

The process by which an RNA polymerase leaves a promoter to become an
efficiently elongating enzyme is poorly understood. In order to better understand
promoter escape, and to characterize the transition from initiation to processive
elongation, methods have been adapted (13, 31, 32) and modified to analyze accurate
RNA synthesis corresponding to the transcription initiation site on adenovirus major late
promoter (AdMLP) in our laboratory. Biotinylated templates are immobilized on
streptavidin-agarose (31, 32), which allows the purification of ternary complexes from
other unbound proteins and nucleotides. In this report, experiments were designed to
study the RNAP H transcription process at the early stage of nascent RNA synthesis

immediately after initiation.

87
MATERIALS AND METHODS

Construction of the Immobilized Templates

The method for preparation of the immobilized template was adapted from
proccedru'es published by other investigators (31, 32). Template DNA containing the Ad-
MLP was synthesized by standard PCR techniques (Perkin-Elmer/Cetus). In PCR
reactions, pBluescriptKSflMLP was the template which includes the sequence of
adenovirus major late (AdML) promoter. Primers were complementary to the region
around -260 and +220, relative to the start point of the transcription. The sequence of the
upstream 5’-biotinylated primer was 5'-biotin-CCCI‘CGAGCGGTG'I'I‘CCGCGGTCCI‘
CC'I‘CG-3', and the sequence of the downstream primer was 5'-CGGTGGCGGCCGCI‘
CTAGAACTAGTGGATC-3'. (Primers were synthesized in the Macromolecular
Structure and Synthesis Facility, Michigan State University). The PCR product was
digested by SmaI endonuclease to generate the 3'-blunt end and passed through a
Sephadex G-50 column to remove the unincorporated biotinylated primer. The purified
PCR DNA was incubated with Streptavidin Agarose (GIBCO BRL) in STE buffer (10
mM Tris [pH 7.6], 1 mM EDTA, 100 mM NaCl) with a rotary mixer at room temperature
overnight. Immobilized templates were washed with STE buffer several times and then
resuspended into H20.
Transcription Assays

An extract derived from HeLa cell nuclei was prepared as described in Shapiro et
a1. (33). HeLa nuclear extract was combined with immobilized DNA and preincubated
for 60 min in microfuge tubes. The pre-incubation reaction volume was 15.2 pl in
addition to the volume of the beads. Reactions were performed at room temperature in 12
mM Hepes pH 7.9, 12 % glycerol, 60 mM KCl, 8 mM MgC12, 3.12 mM EGTA, 0.12
mM EDTA and 1.2 mM D'IT. 10 uU Hexokinase and 50 uM D-glucose were added to
each reaction to deplete ATP and confine transcription initiation to times when a pulse of

nucleoside triphosphates (NTPs) was added. For initiation, 100 uM ATP, GTP, UTP and

88
<1 uM a-[32P] CTP (1o pCi/reaction) or 100 uM ATP, UTP and <1 uM a-[32P] CTP
(10 uCi/reaction) were added and incubated for one minute. Afetr initiation, the tubes
were spun and washed twice by adding 50 til 12 mM Hepes pH 7.9, 12 % glycerol, 60
mM KCl, 3.12 mM EGTA, 0.12 mM EDTA, 1.2 mM DTT, and 1mg/ml bovine serum
albumin. The reaction was either stopped or chased with NTPs. For analysis of short
transcripts, ternary complexes were purified and resuspended in loading buffer (90%
formamide, 1% SDS, 10 mM Tris pH7.9, 1 mM EDTA, and 0.1% bromophenol blue and
0.01% xylene cyanol) and visualized by autoradiography of 23% (20%
polyacrylamide:3% methylene-bis-acrylamide) or composite 8%/23% (top portion of the
gel is 8% and bottom 2/3 is 23%) polyacrylamide gels. For runoff RNA analysis,
reactions were phenol-chloroform extracted and ethanol precipitated. Then, transcripts
were resolved in 6% (30% polyacrylamide:0.8% methylene-bis-acrylamide) or composite
8%l23% polyacrylamide gels and visualized by autoradiography. Transcripts were
quantitated using a Molecular Dynamics Phosphorimager. Details of each experiment are

described in Results and in figure legends.

RESULTS

The stalling of RNA polymerase H can be observed with different radioactive
labeling, and the precise pause is dependent on which NTP is limiting.

An extract derived from HeLa cell nuclei was incubated with. the AdML promoter
immobilized on agarose beads (Figure 1). After formation of preinitiation complexes,
NTPs were added to initiate transcription. One minute after initiation, ternary complexes
were recovered by centrifugation and washing of the beads. Short transcripts were
observed in a one minute pulse before the active elongation on the AdML promoter by
RNA polymerase H, whether labeling is with radioactive CI'P (Figure 2, lanes 2 & 4),
GTP (lane 5) or UTP (lane 6). These short transcripts are sensitive to a-amanitin (lanes

1), and paused complexes were elonagted upon addition of chase NTPs (lane 3).

89

Figure l. Immobilized template: The schematic drawing of biotinylated PCR template
linking to agarose bead through a biotin-Streptavidin linkage.

90

AdML promoter

Agarose bead

+1

 

 

 

 

TATA I" - Smal
TATAAAA CTCACTCI‘CT J
I l l J l

-31 ~25 Inf 2 7

Biotin-Streptavidin linkage

Figure 1

Hpml
from Al
extract 0
bl. Puls:
G‘) at a
C1'13). 1
U‘. M
The rea
Willem
(ll-A) ‘
Marker
ladder

dettrm

91

Figure 2. Synthesis of short transcripts by different labeling of the short transcripts
from AdML promoter. The reaction scheme is shown at the top of the figure. An

extract of HeLa cell nuclei (NE) is mixed with immobilized template and incubate for 1
hr. Pulse NTPs include three unlabeled NTPs at 100 [AM and an a[32P]-NTP (C*, U*, or

G“) at a concentration < 1 uM. Reactions in lanes 1-4 are labeled with C* (a-[32P]
CI'P). The reaction in lane 5 is labeled with 6*, and the reaction in lane 6 is labeled with
U“. After a one minute pulse, complexes are isolated by centrifugation and washing.

The reaction shown in lane 3 is chased for 10 min with 1 mM each unlabeled NTP. The

sequence of AdML transcripts is shown at the bottom of figure. One ug of or-amanitin
(a-A) was added at t = -60 min or during the centrifugation and washing as indicated.
Marker lanes are 5' labeled DNA oligonucleotide A) 18 mer; B) 16 mer and C) oligo dT
ladder (Betheseda Research Laboratories). The precise size of short RNAs are

determined in the experiment shown in figure 4.

92

 

   
 
 

t: +1 min
-60 min t=0 mln Spin & Wash
NE Pulse
Immoblllzed Ad-MLP \ /- Chase
1
hon-A
NE
a-A -60 min
A,C,U,C'
Pulse A,C,U,G‘
A,C,C,U"

Split St Wash +a-A
Spin & Wash - a-A
Chase

 

+236
+20U

+18U

+I7C
+166

+14!)
+13A

‘18mer
16mer

+llG

\
34

  
   

T l 2 3 4 5 6
pppACUCUCUUCCGCAUCGCUGUCUGCG

I
11 14 16 19 22 2!:

 

 

Figure 2

th

sh
ad.
W1
+1

ex;

IlUC
SCVI

imp

lfldk
abor

have

Will
is a 11‘
Figur
Ihe 1i

indie;

93
When a-[32P] CTP is the labeled nucleoside triphosphate, major paused sites are

observed at +llG, +14U and +166, just before the addition of +12, +15 and +17C. Of
all these short transcripts, +1 16 is the most significant one since it contains the most
radioactivity and the fewest labeled C's. Polymerase appaers to be stalled at very similar
positions whether complexes were recovered in the absence or presence of or—amanitin,
indicating that little elongation or exonucleolytic degradation of transcripts occurs during
the purification process (compare lane 4 to lane 2).

When a-[32P] GTP is the radioactive nucleoside triphosphate, the major paused
sites are +18U and +19G (lane 5). Transcription rationally pauses at +18U prior to
addtion of +19G undr the limitation of GTP, whereas pauses at +19G is unexpected.
When a-[32P] UTP is the radioactive nucleoside triphosphate, the major paused sites are
+13A, +17C and +20U (lane 6). The unexpected pause at +20U cannot be simply
explained by limitation of UTP.

Pausing primarily occured at positions that precede addition of a limiting
nucleoside triphosphate, but under less limiting concentration of NTP (Figure 3 ) or under
severe limitation of GTP ( lane 1 of Figure 5) we still observed short transcripts. This
implies that the paussing could be an intrinsic property of the transcription complex.
Transcripts shorter than +llG are barely detectable after washing the complexes,
indicating that such transcripts are not stably associated in the ternary complexes and are
abortively initiated. Similar conclusions about the stability of short ternary complexes
have been made by Luse and colleagues (34, 35).

Stalling of RNA polymerase H is also observed at higher concentration of
radioactive NTP. The primary reason for the stalling of RNA polymerase H is that there
is a limiting concentration of CTP (<1 nM) for the radioactive activity in the reaction. In
Figure 3, we perform an experiment with the a—[32P] CTP and 10 11M CTP. Reducing
the limitation of CTP concentration, we still observe the stalling of RNA polymerase

indicated by the accumulation of short RNAs during a one minute pulse. This result

Figure 3.
The pulse

94

Figure 3. Paused transcripts are also observed in less limitation of radioactive NTP.

The pulse nucleoside triphosphates are 100 M A, G, U and 10 uM C with 10 uCi 0t-
[32P] CI‘P.

.1l.{l|lrr1|.

95

 

 

 

 

 

 

 

 

 

 

l = I min
.NGEo "fin A,C,U,C.+l0HM Spin & WISII
lmmobilizeld AdMLP Stop
a-Amnnitin
NE + + + +
A.G.U,C‘+10 pM C + + + +
or-Amanitin afler Initiation + + + +
Spin & Wash + + + +
l
I +166
,3. _. ,. ‘j +14U
’18.; 1'
' 2" 1 m:
*u *v ‘
pplmfiueULUUEéGéAUéGéUGUéU
l I] I6

 

Figure 3

suggc
RVA

useda

111M}
3'-did¢
dcpem
demon
synthe
of RN,
Since 1
Meter

+1 All

mmSCri
incorpc
lime f
POSitior

inhibiti.

ObSCIVe
recOver
rt-chn

mobillt:

96
suggests that the limitation of the labeling CTP is not the only reason for the stalling of

RNA polymerase H. This result is also true when a-[32P] UTP and 10 uM UTP were
used as the labeling condition (data not shown).

+llG synthesis is dependent upon ATP hydrolysis. One criterion of eukaryotic
mRNA initiation is the requirement for ATP hydrolysis. In Figure 4, we used ddATP (2'-
3'-dideoxy ATP) and AMPPNP (adenosine 5'-B,y-imino triphosphate) to study the
dependence on ATP hydrolysis of +llG synthesis. The results of this experiment
demonstrate that a hydrolyzable B—y bond of ATP or an ATP analog is essential for the
synthesis of +1 10 from the AdML promoter (lanes 1, 2 & 3). As expected, the mobility
of RNA with a 5'-end AMPPNP is different from that of a 5'-end ATP (lanes 1 & 3).
Since AMPPNP and ATP both incorporate into the body of RNA chain as AMP, this
difference is most consistent with initiation of the RNAs with +1 AMPPNP (lane 1) and
+1 ATP (lane 3), as expected for the AdML promoter.

Surprisingly, with the presence of a-arnanitin we observed the elongation of the
transcripts (lane 4). This result suggest that the catalytic sites of RNA polymerase H can
incorporate several nucleotides on the 3'end of RNA in the presence of a-amanitin and
move from +11, +14, +16 to positions at +17 to +19 as well as from +20, +23, +29 to
positions +32 to +34, however the front edge of the emzyes are locked by a-amanitin as
inhibition of transcription.

This 23% gel was run for a longer time to obtain better resolution, and we
observed double bands for each specific signal. These +1 1G-RNA and +14U-RNA were
recovered from the gel slices and digested with calf intestinal alkaline phosphatase then
re-examined on a 23% polyacrylamide gel where they become a single band with a higher
mobility (data not shown). This suggests that there may be a phosphatase activity in the

transcription complex.

Fig
1115
hon.
pres
(ICL
(lane
shon

lanes

97

Figure 4. Short transcripts synthesis are dependent on ATP hydrolysis. ATP,
ddATP (ddA) and AMPPNP (AN) are used to study the requirement for hydrolyzable B—y
bond. AMPPNP can be incorporated in to the first position of the RNA chain in the
presence of ddA (lane 1), but not in the absence (lane 2). Some transcription elongation
occurs in the presence of a-amanitin when 1 mM each chase NTP was added for 1 min
(lane 4; indicated by open arrows). AMPPNP in the first position causes the mobility of
short RNAs to be faster and inhibits dephosphorylation of the 5' triphosphate (compare

lanes 1 and 3).

1 .11 111 1.1 tr .{1 [It

98

 

 

 

 

 

 

 

 

 

 

 

 

 

1: +1 [[1111
Spin 8: Wash
40 min t: 0 min
NE Pulse
Immobilized Ari-MLP /- Chase
1 l l
I I
i :a-A
NE + + + +
II“
Ag!) Ag) 3 (A;
9"” u u u u
C‘ C‘ C‘I C"
a-A alter Initiation + + + 4-
Spin & Wash + a-A + + + '1-
Chase - - - +
,1...
l . +236
. .
g p? +2011
9 .
. g 3 +166
. t, 7
. +141}
1. . I?
"‘ . +110
AMPPNP+116 'C .
l 2 3 4

 

Figure 4

C.-
are deter
of the Db
Also these
properties
hydrolysis
nature of 1
these shor
hansen'pti
ternary c01
iSSigllmen:
ms with
“P99 16d p1
1. initiatioi

ObSCIVCd +

99
Catalytic sites of RNA polymerase H location and precise sizes of short RNAs

are determined by conditional chase. Since the migration of RNA is different from that
of the DNA size marker, the precise sizes of short transcripts are difficult to determine.
Also these short RNAs are characterized as the products of RNA polymerase H using the
properties of a-amanitin inhibition (Figure 2, lane 1) and the requirement of ATP
hydrolysis (Figure 4, lane 1). A direct evidence such as RNA sequence will confirm the
nature of these stalled products. Therefore, we conducted a conditional chase to verify
these short transcripts. In the conditional chase experiment (Figure 5) we first initiate
transcription to allow the synthesis of +116 transcripts, then we spin and wash the
ternary complexes which are assembled on the immobilized templates. Under the size
assignment by which the prominent short RNA is +116, when we supply the appropriate
NTPs with the condition for elongation of transcription we should be able to observe the
expected products according to DNA sequence if the +116-assignment is correct. In lane
1, initiation is under severe limitation of GTP and CTP with no additional chase, we
observed +116 as the major transcript as well as +14U and +166 identically to those in
figures 2, 3, and 4. This +116-ternary complex is chased to +12C-temary complex when
CTP was present during chase (lane 2). When CTP and ATP are added, the +116 is
elongated to +13A (lane 3). When CTP, ATP and UTP are added, the transcript is
elongated to +15C (lane 4). When the elongation NTPs are CI'P, ATP, UTP and 3'-O-
methyl 6TP (m6), we observed a +16m6 with faster mobility (lane5). This +116-
temary complex can only be elongated when the CTP is supplied in the reaction (lanes 6-
10, compare to lanes 1-5 & 11-13). Where there are some newly synthesized +11m6
with higher mobility (lanes 11, 12, 13 & 5) and some transcripts shorter than +116 (lanes
2-5 and lanes 11-13) suggests that these transcripts are generated by S-H mediated
shortening of RNA followed by elongation to the indicated positions. This experiment
directly confirms that these short transcripts are synthesized by RNA polymerase H from

adenovirus major late promoter and these spun-and-washed ternary complexes are

Figur
initiat
comp:
of 1

transc
“11110
hansc

the 10

100

Figure 5. Conditional chase from +116 of the AdML transcript. Transcription is
initiated by a 1 min pulse with ATP, CTP, and a[32P]-CTP. After washing of
complexes, elongation is allowed to proceed with a 1 min chase of various combinations
of 1 mM each CTP, ATP, UTP, and 3'-O-methyl GTP (m6), as indicated. The
transcription extract contained enough contaminating 6TP to elongate to +116 and +166
without addition of GTP to the reaction (lane 1). These data confirm the size of short
transcripts and that short RNAs are initiated from the AdML promoter, also demonstrate

the location of the catalytic site of RNA polymerase H in the ternary complexes.

101

t: 0 min
40 Id- A’U’C'
NE t: +1 min Spin 8: Wash
Immobilized Ari-MLP +/- Chase

 

 

 

i'l‘

+166

+15C
+13A

1113““?

+166 ‘ z : i
9

+141}

' ' +12C

. . +116

+11III6
+1OC

" ‘ " .3 ‘1“:

I" I“. ' - o

l
. .' _.
,, u.»

pppAéUéUéUUééqéAlpépéucuéU

14 16

Figure 5

' {11.1, .

W (;

‘ill '

transcripti
dounstrea
Ru
experimen
that these
autoradiog
short and 1
pulse NTP
condition 1
tmanitin is
are triplicat
Lanes 68 ;
"016d. The
Pre:
pausing pro
in t1’iirlscrip
Varying the
Wished 0 ‘
differences
that all of
Comillexes,
A 1:
c0miller f,
{imitation i:
Washing of
cxrr'ctet is i!

preinitiatiOn

102
transcriptionally active with their catalytic sites locating at +11, +14, and +16 positions

downstream from the transcription start site.

Runoff RNA can be synthesized from the paused ternary complexes. An
experiment with a composite gel (top l/3rd 8%, bottom 2/3rds 23%) provides evidence
that these short transcripts are the precursors of runoff products. Figure 6 is an
autoradiograph of 8%/23% composite polyacrylamide gel that allows examination of
short and runoff RNAs on the same gel. Lanes 1-4 are those reactions with addition of
pulse NTPs only, while lanes 5-8 were prepared identically to lanes 1-4 for the pulse
condition but are elongated with 1 mM each NTP during a 10 min chase protocol. If 0t-
amanitin is added neither short (lane 1) nor runoff (lane 5) RNAs are observed. Lane 2-4
are triplicates of the pulse reaction only. +116, +14 U and +166 transcripts are noted.
Lanes 6—8 are triplicates of elongation reaction with chase NTPs, +217 transcripts are
noted. The +251 transcripts are due to incomplete SmaI digestion of PCR template.

Preinitiation complexes can be isolated by immobilized template. Since
pausing proximal to the AdML promoter might be a process regulated by factors present
in transcription complexes, the stability of the preinitiation complex was tested by
varying the number of washing treatments prior to initiation (Figure 7). Complexes were
washed 0 to 4 times before addition of pulse NTPs without any clearly discernible
differences in the level or distribution of short RNAs (lanes 2 to 5). The results suggest
that all of the factors required for pausing are stably associated with transcription
complexes. Pausing at these positions could be an intrinsic function of polymerase itself.

A 15 to 20 second kinetic lag is observed in formation of stable ternary
complex from the AdML promoter. The kinetics of stable ternary complexes
formation is measured by addition of a-amanitn at various times after initiation and
washing of ternary complexes from adenovirus major late promoter. HeLa nuclear
extract is incubated with immobilized AdML template for one hour to allow the

preinitiation complexes to form. At t = 0 min, A, 6, U and C“ are added to initiate the

Figu;
a 8‘}
tranSt
positi
SmaI

comp

103

Figure 6. Short transcripts are the precursors of runoff RNA. An autoradiograph of
a 8%/23% composite gel allows the comparison between short RNAs and runoff
transcripts. Short transcripts (lanes 24) can be chased quantitatively to the +217 runoff
position (lanes 68). Some PCR-synthesized templates are not efficiently digested by
Smal generating a +251 runoff position. Isotope that accumulated at the interface of the

composite 8% and 23% gel (open arrow) does not represent a specific AdML transcript.

104

 

1: +1 min
so min t: 0 min Spin & Wash
. .
NE A,C,U.C
lmmobllizfid-MLP 7' Chase]
A Ara-A
NE + + + + + + +
a-A -60 min +
A.G,U,C" +
Spin & Wash +
Chase
25]
8% gel 217
23% gel +166
+14U
+1 16

 

 

 

Figur 6

Fig

heft

105

Figure 7. Washes of the preinitiation complexes do not alter the stalling of RNA
polymerase H. Different number of washes are applied to the preinitiation complexes

before initiation in lanes 3, 4, and 5 compare to no wash treatment in lane 2.

106

 

Pulse
t: 0 min AthU’C'
-60 ml Spin 8; Wash of
NE 11 preinitiation complexes Spin & Wash
Immobilized Ad-MLP Stop
1 1
+/- a-A
NE
a-A -60 min
Washes before Pulse
AQG’U9C.
Spin & Wash
+236
+20U
+166
+14U
+116

 

 

 

 

Figure 7

syntht
the re
transc
were
plotte.
+1 16
the se
hydro
preini
pulse
ternar
unafft
Whicl
from
sugge
bond

betwt

107
synthesis of short RNAs. At t = 0, 5, 10, 20, 40 and 60 seconds, a-amanitin was added to

the reactions. After spin and wash, stable ternary complexes were extracted and short
transcripts were analyzed on a 23% polyacrylamide gel. The prominent +116-transcripts
were quantitated using a Molecular Dynamics Phosphorimager and quantitation were
plotted as Figure 8. Surprisingly, a 15 to 20 second lag precedes the accumulation of
+116 (filled squares, I). ATP hydrolysis has been suggested as an energy source during
the separation of template strands prior to phosphodiester bond formation in RNA. ATP
hydrolysis would also allow for phosphorylation of the CTD on polymerase H in the
preinitiation complex. Therefore, ATP was added one minute before the addition of
pulse NTPs to determine whether this treatment will shorten the observed lag in stable
ternary complexes formation which is precursor of runoff RNA. However, the lag is
unaffected (open circles, 0). Even addition of ATP and a-[32P] CTP (filled circles, O),
which provides for both ATP hydrolysis and formation of the first phosphodiester bond
from the adenovirus major late promoter, does not suppress the kinetic lag. These results
suggest that the lag is not due to either open complex formation, or to first phosphodiester
bond formation, or to phosphorylation of CTD on polymerase H. The slow step occurs
between formation of the first and tenth phosphodiester bonds.

RAP74 contributes to the stability of ternary complexes. We have previously
shown that RAP74 is required for promoter escape (20). One way in which RAP74
might be involved in this process could be to stabilize short transcripts so that they are
not released from polymerase. Short transcripts initiated in the presence of full length
RAP74 are very stable to washing and can quantitatively be elongated to a runoff
position. Since RAP74 mutants have different efficiencies in a runoff transcription assay
(37), these mutants were tested to determine whether the yield of runoff products was
affected by initiation efficiency or by the stability of ternary complexes.

An extract derived from the nuclei of HeLa cells was depleted of RAP30 and

RAP74 by immunoprecipitation with anti-RAP30. Such an extract is completely

iigu ['1
synthe
stable
Quan‘
Show
kinetf
Sure 1.
(Opel
Prior

obse

108

Figure 8. A 15 to 20 seconds lag in stable ternary complexes synthesis. RNA
synthesis was inhibited by addition of 1 mg/ml a—amanitin (or-A) at the times indicated,
stable ternary complexes were isolated , and the +116 AdML transcript was quantitated.
Quantitation of total short transcript synthesis was qualitatively very similar to that
shown for +116, and all short AdML RNAs accumulated with approximately the same
kinetics (data not shown). 0, 5 and 10-second time points were done in triplicate to be
sure that low signals were not due to experimental error. Addition of 100 11M ATP alone
(open circles, O),Or 100 mM ATP + <1 ttM a32[P]CTP (filled circles, O), for 1 min
prior to addition of pulse ATP, GTP, UTP, and a[32P]CTP, did not suppress the

observed lag.

109

 

 

 

 

 

 

100
z ”W
o /‘
g ,t’ I AGUC‘
3 >‘ {5" o A-i-AGUC‘
E. 3 so - ,t‘ o AC‘+AGUC*
a: >
g i
U V
O
<
Time (s
-60 min )
NE t= 0 min
Immobilized Ad-MLP A,C,U,C*
l I I
Add or-A at
0, 5, 10, 20, 40 and 60 s

Figure 8

depe

lengt
end

stab]
triph

and

0118

and

that

RA]

equi

obte

diff:

1621C

mp

517

difl

aitr

was
but

Syn

110
dependent on addition of RAP30 and RAP74 to restore accurate transcription. Full

length RAP74 (1-517), and a set of mutants deleted progressively from the C—terminal
end were tested for the ability to support accurate initiation and for the ability to form
stable ternary complexes. In previous work, using «[32P]-6TP as the limiting nucleoside
triphosphate, C-terminal mutants showed an ever-decreasin g efficiency of transcription,
and mutants deleted beyond 1-205 showed no ability to produce a runoff transcript (37).

The template for transcription is the Adenovirus major late promoter immobilized
on agarose beads. Transcripts were initiated with a 1 min pulse with 100 11M ATP, UTP,
and GTP, and <1 11M a[32P]-CI'P. In Figure 9B lanes 1-3, transcripts were immediately
chased with a mixture of 1 mM ATP, CI‘P, UTP and GTP. In single round transcription,
RAP74 1-517, 1-205, and 1-172 each supported initiation and runoff transcription with
equivalent efficiency. The apparent discrepancy between this result and that previously
obtained in which the 1-17 2 mutant appeared to be inactive may be attributable to the
different nucleoside triphosphate concentrations used in the transcription protocols. The
position of pausing is dependent on the nucleoside triphosphate that is limiting in the
reaction, and the site of pausing may determine transcript stability.

In lanes 4-6, complexes were washed twice with buffer before chase nucleoside
triphosphates were added. In this case, the results are much more comparable to those
previously obtained with a GTP-limited reaction. The complex initiated with RAP74 1-
517 is efficiently elongated to the runoff position. The 1-205 mutant makes a runoff
product with much lower efficiency, and the 1-172 mutant makes very little product. The
different efficiencies of these mutants for production of the runoff RNA may be
attributable to the stability of the initiated complexes.

Short RNA products initiated with these mutants are visualized in Figure 9C after
washing. These transcripts were initiated in a 1 min pulse as described above, washed
twice, and then visualized on a 23% polyacrylamide gel. Since these transcripts are

synthesized under conditions of CTP limitation, the product observed is primarily

wit
30(
ten

phi

Sta
mt
Wa
R}

Sta

111

Figure 9. RAP74 is important for the stability of initiated transcription complexes.
A) RAP74 deletion mutants. B) Sequences between 205 and 517 and between 172 and
205 can contribute to the efficiency of runoff transcription. Transcription was from the
Adenovirus major late promoter immobilized on agarose beads. An extract derived from
human HeLa cell nuclei was depleted of RAP30 and RAP74 by immunoprecipitation
with anti-RAP30 antibodies. This extract (7 111) was combined with 150 ng RAP30 and
300 ng RAP74 1-517 or RAP74 deletion mutant and incubated with immobilized
template for 60 min. Initiation was by addition of 100 M ATP, UTP, and GTP, and <1
le a[32P]-CI'P for 1 min. Reactions shown in lanes 1-3 were immediately chased with
1 mM ATP, CTP, UTP, and GTP (no wash). Reactions shown in lanes 4-6 were washed
twice with 50 1.11 of wash buffer before addition of chase nucleoside triphosphates (wash).
C) Inspection of short RNAs reveals RAP74 sequences that contribute to ternary complex
stability. Short RNAs were initiated in the presence of RAP74 1-517 or RAP74 deletion
mutants in a 1 min pulse as described for B above. Immobilized ternary complexes were
washed twice with buffer and short RNAs were visualized on a 23% acrylamide gel.
RAP74 sequences between 409 and 517 and between 172 and 205 contribute to the

stability of short ternary complexes.

112

17

5

 

 

 

 

205

172

 

l

 

 

Figure 9

1 13
elongated to +116. The transcript initiated with RAP74 1-517 is most stable. Deletion to

1-409 or 1-205 reduces stability somewhat. A further decrease in ternary complex
stability is seen with deletion to 1-172, as predicted from the data shown in Figure 9B
lanes 5 and 6. The conclusion of these experiments is that RAP74 mutants as short as l-
172, and possibly shorter, support accurate initiation as well as full length RAP74.
Ternary complexes formed with these RAP74 mutants, however, have varying stability.
Apparently, sequences between 409 and 517 and between 172 and 205 contribute to the
stability of short ternary complexes. This observation is very consistent with the role of

RAP74 in promoter escape.

DIE

cor.
hyc'

for

161']
len
init

the

mi
Al
fln

5121
1111
eff
Co
cer
mu

dif:

1 14
DISCUSSION

Using an immobilized template assay, we have observed the first stable ternary
complexes accurately initiated from the AdML promoter. Initiation requires ATP
hydrolysis and separation of DNA template strands (39, 40). Once phosphodiester bond
formation commences, however, RNA polymerase H must synthesize a chain of a
minimum length to form a stable complex (13). Consistent with this idea, no stable
ternary complexes shorter than +116 were detected indicating that this is the minimal
length for stability (Figure 2). RNA polymerase H pauses transcription shortly after
initiation. Varying the nucleoside triphosphate that was limiting in concentration dictated
the tendency to pause at particular sites, and in most cases, polymerase stalled at
positions preceding addition of the limiting NTP. Synthesis of short RNAs required
hydrolysis of ATP and was completely eliminated by addition of a-amanitin. Paused
RNAs were quantitatively chased to the expected runoff position with addition of chase
NTPs. Short, paused RNAs accumulated after a 15-20 s delay, indicating that this is the
minimal time required to synthesize the +116 transcript. Pre-incubation with ATP, or
ATP and CTP did not eliminate this delay, indicating that open complex formation and
first phosphodiester bond formation do not account for the delay in stable complex
synthesis. The delay appears to represent the time required to form phosphodiester bonds
between addition of +3U and +116.

By analysis of mutants, the RAP74 subunit of TFIIF was shown to be required for
stability of these short transcripts. RAP74 is a protein of 517 amino acids. Deletion
mutants 1-409, 1-205, and 1-172 were all shown to initiate transcription with equivalent
efficiency to the full-length protein, but did not support complexes of equivalent stability.
Complexes formed in the presence of the full length protein showed no sensitivity to
centrifugation and washing. A subset of complexes formed in the presence of the 1-409
mutant were stable but a fraction were disrupted by centrifugation and washing. No

differences were noted in stability between the 1-409, 1-356, 1-296 and 1-205 mutants.

Set

pro
on
mu

5121

C0

812

It

115
Sequence between 409-517 appears to be required for stability of a subset of complexes

(about 60%) but not for all. The 1-172 mutant showed lower stability than these longer
proteins, although this mutant showed equivalent initiation efficiency. In most cases, two
washes completely dissociated the ternary complex formed in the presence of the 1-172
mutant, but in some experiments this mutant has been observed to have some ability to
stabilize these complexes (Figure 9B, lane 3, and data not shown), so part of the stability
region remains intact in the 1-172 mutant. Essentially all of the newly-initiated
complexes appear to require sequence between amino acids 136-205 of RAP74 for
stability. Thus, there appear to be at least two distinct classes of elongation complex that
may contain slightly different constellations of associated transcription factors. One class
requires C-terminal sequences between 409-517. The Other class requires sequence
between 1-205 but not C-terminal sequences. It is likely that the class that requires C-
terminal sequences also requires the N-terminal region, but this point cannot clearly be
made by our experiment.

The observation that RAP74 stabilizes short ternary complexes is very consistent
with results previously published by our laboratory in which we showed that RAP74
could be completely dispensible for accurate initiation in an extract transcription system
(20). Evidence for RAP74-independent initiation was obtained using two methods: 1)
RNA was pulse-labeled in the absence of RAP74; and 2) sarkosyl-resistant complexes
were formed in the absence of RAP74. In both cases, however, RAP74 was required to
elongate the RNA. This result has been somewhat controversial in the literature,
however, because RAP74 can clearly contribute to initiation functions. RAP74
contributes to the stability of pre-initiation complexes in polyacrylamide gel mobility
shift assays (19). RAP74 also must incorporate into a complex consisting of TBP, TFHB,
RAP30, and RNA polymerase H, for RAP30 to cross-link to DNA (41). Normal
assembly of RAP30, therefore, appears to require RAP74. In another report, both RAP30
and RAP74 were required to produce runoff products and short RNAs (42). In our own

on;

ABC

1111

pt:

Dr
Wt

ab

fa

l 16
unpublished data, we had similarly observed that in some extract systems, both RAP30

and RAP74 were required to initiate and recover stable transcripts.

To reconcile these apparently inconsistent observations, we speculated that
initiation by RNA polymerase H did not require the RAP74 subunit of TFHF, as we had
previously observed (20). We further considered that other unidentified factors, present
in some systems but absent from others, could stabilize ternary complexes and prevent
release of short RNAs from polymerase. Such a stabilizing factor would be similar to
Drosophila p-TEF (positive transcription elongation factor) described by Price and co-
workers (32). In the presence of such a factor or factors, initiation would occur in the
absence of RAP74 and the ternary complex would remain stably associated with
polymerase. Addition of RAP74 would still be necessary for early elongation because
RAP74 is required for promoter escape (20). In a system missing such a stabilizing
factor, RAP74 will appear to be required for initiation, but the requirement in this case is
to stabilize the ternary complex (Figure 9).

A model is presented to describe the functions of RAP74 in accurate initiation and
promoter escape from the AdML promoter (Figure 10). This model has features that are
consistent with other models recently presented for RNA polymerase H elongation and
"inchworming" (38), and this model can reconcile our and other's experimental results for
TFIIF function. RNA polymerase H is indicated as an oval with two structures that
interact with RNA, the catalytic center and an RNA binding site (RBS). The catalytic
center is the site at which phosphodiester bonds are formed. The RBS is a site at which
RNA is tightly bound, but through which RNA translocates during elongation.

Abortive initiation occurs when polymerase synthesizes RNAs that are too short
to occupy RBS (<+116). Such RNAs are released, probably without dissociation or
movement of polymerase, and re-initiation can subsequently occur (13, 35). Synthesis of
short RNAs (+116 or longer) fills RBS, and this process requires 15-20 s (Figure 8).
Occupancy of the RBS is the step designated promoter escape I. The complex that is

117

Figure 10. A model describing the role of RAP74 in accurate transcription and promoter
escape by RNA polymerase H. The catalytic center of RNA polymerase H is the site of
phosphodiester bond formation. RBS is an RNA binding site on polymerase through

which the chain must translocate. See the text for details.

118

@ catalytic center open complies
—> I335]
RNAP H abortive initiation

 

 

 

 

 

 

 

 

L ' abortive RNAs
ppl>~/
RAP74 -
—( ) <+llG
?_+, :1,
?—-(+) ‘promoter escape I
r z+llG
homily-initiated] complexes
‘ +l7C - +196

 

       

RAP74 —-(+)
a-amanitin ,_(_) promoter escape II

C P +20U
g .

 

 

 

 

 

 

  

 

 

 

PPP
PPP—
a-amanitin —(-) *
C , +35U
PPPV— \l—__r

 

Figure 10

119
formed is. similar to those previously described as initiated or sarkosyl-resistant

complexes (20). Goodrich and Tjian (1994) have shown that TFIIE and mm are
required for promoter escape I, but our data demonstrate that the RAP74 subunit of TFIIF
can be dispensible for this step (43). Other factors that have not been clearly identified
enhance stability of the complex.

Particular sequences within RAP74 are required to preserve these complexes. The
sequence between amino acids 409-517, at the very C-terminus of RAP74, is involved in
making contacts with RNA polymerase H (B.Q. Wang and Z.F. Burton, submitted),
TFIIB (S.M. Fang and Z.F. Burton, in preparation), and DNA (B.Q. Wang and Z.F.
Burton, submitted). RAP74 sequence between 172 and 205, which appears to be critical
for stability, is involved in binding to the RAP30 TFIIF subunit.

"Inchworming" by RNA polymerases has been described as a shift in the distance
between two RNA interaction sites (38). This can be drawn as a change in polymerase
conformation or, as indicated here, as the RNA distance between the catalytic center and
the RBS. According to either view, polymerase reaches boundaries to elongation at
which a transition within the enzyme must occur for synthesis to continue. This
transition involves shortening the RNA length between the catalytic center and the RBS
and translocation of RNA through the RBS.

Our data indicate that RNA polymerase H may undergo two inchworrn transitions
as it escapes the AdML promoter. When a-aminitin is added with NTPs to initiated
complexes, elongation proceeds to two distinct boundaries near +196 and +34C. 0t-
amanitin, therefore, allows phosphodiester bond formation, but blocks the inchworrn
transition and chain translocation, as indicated in the figure. According to this view, a-
amanitin blocks a conformational change in polymerase that is required to decrease the
RNA distance between the catalytic center and RBS, or blocks RNA translocation

through the RBS, or both.

120

This same transition that is sensitive to a-amanitin may be promoted by TFIIF,
since RAP74 is required for early elongation from the AdML promoter (20). This step is
designated promoter escape II. Promotion of similar transitions during many steps in
subsequent synthesis could explain how ‘I'FIH2 stimulates elongation rates (44).

ACKNOWLEDGEMENTS

This work was supported by the National Institutes of Health, The Michigan State
University Agricultural Experiment Station, and Michigan State University.

121
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10. Levin, J. R., Krummel, B., and Chamberlin, M. J. ( 1987) Isolation and properties of
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11. Krummel, B., and Chamberlin, M. J., (1989) RNA chain initiation by Escherichia
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12. Gill, S.C., Weitzel, S.E. and von Hippel, RH. (1991) Escherichia coli 070 and NusA

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13. Luse, D.S. and Jacob, G.A. (1987) Abortive initiation by RNA polymerase H in vitro

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14. Horwitz, R. J., Li, J. and Greenblatt, J. (1987) An elongation control particle

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l6. Buratowski, S., Hahn, S., Guarante, L., Sharp, P. A. (1989) Five intermediate

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17. Maldonado, E., Ha, I., Cortes, P. Weis, L., Reinberg, D. (1990) Factors involve in

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18. Killeen, M. and Greenblatt, J. (1992) The general transcription factor RAP30 binds

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19. Flores, 0., Lu, H., Killeen, M., Greenblatt, J., Burton, Z.F. and Reinberg, D. (1991)
The small subunit of transcription factor IIF recruits RNA polymerase II into the
preinitiation complex. Proc. Natl. Acad. Sci. USA 88, 9999-10003.

20. Chang, C.-h., Kostrub, C. F. and Burton, Z. F. (1993) RAP30/74 (Transcription
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21. Corden, J.L. (1990) Tails of RNA polymerase H. Trends BioL Sci. 15, 383-387.

22. Flores, 0., Lu, H. and Reinberg, D. (1992) Factors involved in specific transcription
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23. Lu, H., Zawel, L., Fisher, L., Egly, J .-M. and Reinberg, D. (1992) Human general
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24. Schaeffer, L., Roy, R., Humbert, 8., Moncollin, V., Vermeulen, W., Hoeijmakers, J.
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27. Lis, J. and Wu, C. (1993) Protein traffic on the heat shock promoter: parking, stalling
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28. Spencer, CA. and Groudine, M. (1990) Transcription elongation and eukaryotic
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29. Giardina, C., Perez-Riba, M. and Lis, J.T. (1992) Promoter melting and TFIH)
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HeLa cell nuclear transcription extract. DNA 7, 47-55.

34. Jacob, G. A., Luse , S. W., and Luse, D. S. (1991) Abortive initiaiton is increased
only for the weakest two members of a set of down mutants of the Adenovirus 2 major
late promoter. J. Biol. Chem. 266, 22537-22544.

35. Jacob, G. A., Kitzmiller, J. A., and Luse. D. S. (1994) RNA polymerase H promoter
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36. Goliger, J. A., and Roberts, J.W. ( 1989) Sequence required for antiternrination by
phage 82 Q protein. J. Mol. Biol. 210, 461-471.

37. Wang, B.Q., and Burton, Z. P. (1995) Functional domains of human RAP74
including a masked polymerase binding domain. Submit to J. Biol. Chem.

38. Chamberlin, M. J. (1995). New models for the mechanism of transcription
elongation and its regulation. Harvey Lect. 88, 1-21.

886 Bunick, D., Zandomeni, R., Ackerman, S. and Weinmann, R. (1982). Cell 29, 877-
40. Sawadogo, M. and Roeder, R. 6. (1984). J. Biol. Chem. 259, 5321-3526.

41. Coulombe, B., Li, J ., and Greenblatt, J. (1994) Topological localization of the human
transcription factors HA, IIB, TATA box binding protein, and RNA polymerase H-
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123

42. Tan, 8., Aso, T., Conaway, R. C., and Conaway, J. W. (1994). Roles for both the
RAP30 and RAP74 subunits of transcription factor HF in transcription initiation and
elongation by RNA polymerase H. J. Biol. Chem. 269, 25684-25691.

43. Goodrich, J. A. and Tjian, R. (1994). Transcription factors HE, IIH and ATP
hydrolysis direct promoter clearance by RNA polymerase H Cell 77, 145-156.

44. Bengal, E., Flores, O., Krauskopf, A., Reinberg, D. and Aloni, Y. (1991). Role of
the mammalian transcription factors HF, IIS, and IIX during elongation by RNA
polymerase II. Mol. Cell. Biol. 11, 1195-1206.

CHAPTER IV

YEAST Cdc73p MAY BE A
COMPONENT OF AN ALTERNATE VERSION OF TFIIF

124

125
ABSTRACT

Cdc73p was isolated using a monoclonal antibody directed against the carboxy
terminal domain of RNA polymerase H. Comparison of Cdc73p sequence with that of
human RAP30 and bacterial sigma and delta transcription factors led to the idea that this
diverse collection of proteins might have similar functions. We propose the model that
Cdc73p is a subunit of an alternate form of TFHF, that functions in transcription of an
alternate promoter class recognized by RNA polymerase H, in much the same manner
that bacteria utilize different sigma factors to transcribe from alternate promoters. This
model makes many predictions that are subject to experimental test. One prediction is
that Cdc73p will interact directly with RNA polymerase 11 through a conserved
polymerase binding region. Consistent with this prediction, Cdc73p binds to RNA
polymerase H. Cdc73p appears as an additional polymerase subunit, when polymerase is
separated from free Cdc73p by gel filtration. Cdc73p sequences within the predicted
polymerase interaction region are critical for binding. Another prediction of this model is
that Cdc73p may interact with ng3p/Anclp, the smallest subunit of yeast TFIIF. In a
preliminary experiment, this expectation appears also to be correct. Several other tests of

this model are in progress, and these approaches are discussed.

126
INTRODUCTION

The yeast gene CDC 73 was initially identified in a suppressor screen of ste2
deletion mutants that are defective in mating. Ste2p is the a factor receptor. Binding of
or factor to Ste2p on a type cells stimulates a signal transduction cascade that results in
cell cycle arrest at Gl/S (1). Cell cycle arrest is necessary prior to fusion of a and or type
cells to create alert diploids.

The CDC designation stands for cell division cycle, and temperature sensitive
cdc73 mutants arrest cell division when shifted to the non-permissive temperature,
although it appears that arrest occurs at 62/M rather than at Gl/S (2). CDC73 does not
appear to function directly in or factor signalling, and its participation in mating may be
indirect, perhaps through its function in regulating the cell cycle.

Transcription factor TFIIF in yeast is composed of three subunits named
nglp/Ssu71p, ng2p, and ng3p/Anclp (3). In humans TFIIF is composed of two
distinct subunits, RAP74 which is homologous to nglp/Ssu71p, and RAP30 which is
homologous to ng2p. TFIIF is required for accurate initiation by RNA polymerase H
and stimulates elongation of transcription (4, 5). TFIIF assists polymerase binding to the
promoter and so this factor is at the core of the assembly pathway for an active pre-
initiation complex (6). Because of its essential role in polymerase enu'y, TFIIF must join
the complex before TFIIE and TFIIH can stably enter. To a large extent, TFHF directs
the final assembly of the transcription complex.

Human RAP30 is related structurally and functionally to bacterial sigma factors.
(7, 8) Our analysis indicates that RAP30 may have sequence similarity to conserved
regions 1.2, 2.1, 3.1, and 4.1 of bacterial sigma factors and a region of bacterial delta
factor (9). Region 2.1 is the most important sequence for polymerase binding by sigma
factors, and mutagenic analysis shows that this is the polymerase binding region of

RAP30 as well (10, 11). The C-terrninal region of RAP30 contains a masked DNA-

127
binding domain, just as do similar regions 3.1 and 4.1 of sigma factors (12, 13). RAP30

alters template contacts by polymerase in a manner analogous to bacterial sigma factors,
and the functions of these proteins are similar in directing polymerase to the promoter.
Yeast RNA polymerase H has 27 repeats of the heptapeptide consensus YSPTSPS
at the C-ternrinus of the largest subunit (14). Using affinity chromatography with
antibody directed against this carboxy terminal domain (CTD) as a leash to capture RNA
polymerase 11, our laboratory identified TFIIF subunits and Cdc7 3p as polymerase
associated proteins (15). Based on a sequence comparison between Cdc73p, human
RAP30, yeast ng2p, and bacterial sigma factors, we concluded that yeast Cdc73p was
likely to be a subunit of an alternate form of TFHF (9). Cdc73p appears to be similar in
structure to bacterial sigma factors within conserved regions 1.2, 2.1, and 3.1. Cdc73p
also has an extended similarity to bacterial delta factor (9). In this report, we have

subjected this model to some preliminary experimental tests.

128
MATERIALS AND METHODS

Expression vector and strains. T7 expression plasmid pET21a and expression
strain BL21(DE3) were purchased from Novagen. Manipulation of the expression
system was according to manufacturer's instructions and has been described previously
(16).

The CDC73 gene was amplified by a standard PCR reaction. The 5' primer (5'-
CATATGGC6AACTCAT1‘A6ACA6ACTGAGA6—3’) was designed to create an Nde I
site at the position of initiator methionine. The 3' primer (5'-GCGGCC6CACGGTATCC
TCT'I‘GAAATAAGTTCC—3') was designed to create an Not I site to fuse the CDC73
open reading frame to a sequence terminated with six histidines. The Nde I to NotI
fragment was cloned between the Nde I and Not 1 sites of the pET21a vector.

The internal deletion mutant, cdc73pA129-l43, was generated as follows. The
first 128 amino acids of Cdc73p using 5'-primer as above and the following primer 5'-
CT CGAGGTCT CGCCT'I'I‘ CI‘ GACCGGGTGCTT -3' which introduces an XhoI site at
the end of the first 128 amino acids of Cdc73p. This NdeI-Xhol fragment was cloned
into pET21a/CDC73 which had been previously digested by NdeI and SalI, preserving
the reading frame and precisely deleting amino acids 129-143.

The C-terminal deletion mutant, cdc73p1-143 was created by digesting the
pET213/CDC73 with restriction enzymes SalI and XhoI, filling in with Klenow enzyme
and religating.

Production and purification of Cdc73p, cdc73p A129-l49 and cdc73p 1-143.
Bacterial cultures were grown up to optimal density, induced with IPTG and harvested as
described previously (17). Proteins were purified by N12+ affinity chromatography
purchased from QIAGENE, using a previously described protocol (17).

Production of anti-Cdc73p antiserum. Antisera against Cdc7 3p was produced

in rabbits. Titer Max (Ctny) was included as the adjuvant in the initial injection but not

129
in the following booster injections. The protocol is essentially the same as that

previously used for production of anti-RAP30 and anti-RAP74 antiserum (17).

Gel filtration chromatography binding assay. A three-fold molar excess of
Cdc73p, or A129-143, or 1-143 were mixed with purified yeast RNA polymerase H in 0.5
M KCl storage buffer and centrifuged through an Amicon filter to concentrate the sample
to a volume smaller than 200 111. Samples were dialyzed against 0.1 M KCl storage
buffer (20 mM Hepes pH 7.9, 20 % glycerol, 0.5 M KCl, 0.2 mM EGTA, 0.2 mM EDTA
and 2 mM DTT) overnight at 4°C to allow binding. A Superdex 200 HR 10/30 gel
filtration column purchased from Pharmacia was equilibrated with 0.1 M KCl storage
buffer (20 mM Hepes pH 7.9, 20 % glycerol, 0.1 M KCl, 0.2 mM EGTA, 0.2 mM EDTA
and 2 mM DTT) at a flow rate of 0.5 ml/min until a stable base line was reached.
Samples were cleared by brief centrifugation before injection. Peaks from elution
profiles were collected and spin concentrated before loading on SDS PAGE. Proteins
were visualized by Coomassie blue staining.

Anclp binding assay. Anclp (120 11g) was immobilized on Affi-gel 10 (Pierce)
as reported by Lei and Burton (18). Non-specific binding was blocked by incubating the
immobilized Anclp and control resin with reaction buffer containing 20 mM Hepes, pH
7.9, 20 % glycerol, 0.5 M KCl, , 0.2 mM EGTA, 0.2 mM EDTA, 2 mM DTI‘ and 0.2%
BSA at 4°C for one hour. Cdc73p (20 11g) was added and the sample incubated for
another 30 rrrin. The beads were collected and washed 5 times with the same reaction
buffer but without BSA. Bound Cdc73p was eluted with 40 111 0.5 M KCl reaction
buffer. The eluates were analyzed by SDS PAGE, and proteins were stained by silver

staining.

130
RESULTS

Cdc73p is associated with yeast RNA polymerase II. A yeast whole cell
extract was fractionated by affinity chromatography on a column containing covalently
immobilized monoclonal antibodies directed against the CTD of RNA polymerase H
(19). This antibody captures polymerase along with a complex set of associated proteins
(RAPs), which can be dissociated from the core enzyme by treatment with moderate salt
(ie. 0.5 M KCl). A negative control column contains an immobilized monoclonal
antibody of the same isotype (Ig62a), directed against a protein not found in yeast (15).
Initially, RAPs were identified as those proteins that bind to the anti-CTD column but not
to the negative control column. The arrrino acid sequence of an internal peptide derived
from a RAP of approximately 54 kDa was identical to a sequence found within the
coding frame of a previously identified yeast gene CDC73. The selectivity of Cdc73p
binding to the anti-CTD column is confirmed in the experiment shown in Figure 1. The
0.5 M KCl eluates of the anti-CTD and anti-[3' columns are compared in a Western blot
developed with anti-Cdc73p antibodies. Cdc73p is detected in the eluate of the anti-CID
column (lane 2) but not in the eluate of the anti-[3' column (lane 3). The recombinant
Cdc73p-H6 marker appears slightly larger than the normal yeast protein because of the C-
terminal polyhistidine extension.

Cdc73p may share sequence similarity to human RAP30 and bacterial 0
factors within their RNA polymerase binding regions. Inspection of the amino acid
sequence of Cdc73p indicated that this protein might be evolutionarily related to human
RAP30 and bacterial sigma factors within the regions of these proteins that binds to their
respective RNA polymerases (9). In Figure 2, a proposed alignment of yeast Cdc73p
with the RNA polymerase H binding region of human, Xenopus, and Drosophila RAP30,
and the RNA polymerase binding region of bacterial sigma factors is shown.

Hydrophobic cluster plots of the sequences used in the alignment are also shown.

131

Figure l. Cdc73p is associated with yeast RNA polymerase H. A yeast whole cell
extract that is active in accurate transcription was fractionated over columns containing
covalently immobilized monoclonal antibodies directed against the CTD of RNA
polymerase II or against the E. coli RNA polymerase subunit B'. Yeast RNA polymerase
H and associated proteins were immobilized by binding to the anti-CTD antibody. These
proteins were not expected to bind to the column containing anti-[3' antibody. The 0.5 M
KCl eluates of the anti-CTD column (or-CTD) and the negative control column (or-B') (80
1.11 each) were analyzed in a Western blot developed with anti-Cdc73p antibodies. 100 ng

of recombinant Cdc73p-H6 (lane 1) is shown as a reference standard.

132

.QIRV
QEUAV

E355

 

 

Figure 1

133

Figure 2. Cdc73p may share sequence similarity to human RAP30 and bacterial 0
factors within their RNA polymerase binding regions. At the bottom of the figure are
hydrophobic cluster analysis plots of polymerase binding regions of human and

Drosophila RAP30, and two bacterial sigma factors, sigma A (Anabena) and sigma 70

(E. coll). These plots are the basis for the alignments shown above. This analysis was

performed by Shi Min Fang.

134

requiredforpolllbinding

I

hRAP30 142 SQQLDKVVTTNYKPVBNHQYN.IEYERKKKEDGKR 176

XRAP30 156 SQQLEKAVTSNYKPVSNHQYN.IEYEKKKKDDGKR 190

dRAP30 158 VQPIDKIVLQNFKPVKDHAHN.IEYRERKKREGKK 192
it *tlt * at or ' *t t 1*

Cdc73p 131 VDIQNKTLAQELSTVKSTTSASLEHDSEVSDPVVE 165
l !

 

required for pol H binding
sigma70 RBAKDKMVQSNLRLVVSLAKK.YMHRGLSEQDLIQ
sigmaA RBAKDEMVQSNLRLVVSLAKK.YMHRGLSEQDLIQ
sigmaG DSAREKLVNGNLRLVLSVIQR.FNMRGEYVDDLFQ
sigmaF QQARDLLIEKNNRLVWSVVQR.ELNRGXEPDDLEQ
sigmaH SDALDXLITKYRNFVRAKARS.YELIGADREDIVQ

.—

(Bold indicates match to >50% of consensus of sigma factors, underline indicates 30-
50%, and italic indicates 10-30%, calculated from 31 sigma factors. Stars indicate
conserved residues between Cdc73p and RAP30.)

 

Figure 2

 

135
Additional sequence similarities between these proteins (not shown) further suggested

that these alignments might be correct.

Production and purification of Cdc73p and Cdc73p mutant proteins. Binding
to RNA polymerase II detected by anti-CID chromatography (Figure 1) might either be
direct or indirect. If Cdc73p includes a polymerase binding region, as we propose,
Cdc73p should bind directly to RNA polymerase H. Our sequence alignment further
predicted that binding would be sensitive to mutation within the proposed polymerase
binding domain. Two cdc73p mutant proteins were constructed to determine the
importance of this sequence for binding (Figure 3). Mutant cdc73pAl29—l43 has a 15
arrrino acid deletion within the pr0posed polymerase binding site, so this mutant was
expected to exhibit decreased binding affinity. Mutant cdc73p1-143 has most of its
polymerase binding domain intact, but the C-terminal sequences are deleted. The
production and purification of Cdc73p and Cdc73p mutant proteins is shown in Figure 4.
The cdc73p1-143 mutant was expected to bind RNA polymerase H because this mutant
was previously shown to have partial function in vivo (1). Complete truncation of
CDC73 is lethal and the 1-143 truncation is temperature sensitive.

The predicted interaction region of Cdc73p is important for RNA
polymerase II binding. Binding to RNA polymerase H was tested using a gel filtration
protocol. Yeast RNA polymerase H is a large complex of 10 distinct subunits with a
molecular weight of approximately 500 kDa. Cdc73p at 44 kDa appears to be monomeric
by gel filtration and is easily separated from RNA polymerase H (data not shown). In
Figure 5, Cdc73p and cdc73p mutants were tested for polymerase binding. RNA
polymerase H was combined with a three fold molar excess of Cdc73p or cdc7 3p mutant
in buffer containing 0.5 M KCl. Since this buffer was known to release Cdc73p from an
anti-CTD column, binding was not expected under this condition, but the solubility of the
recombinant proteins was maintained. Binding reactions were then concentrated by

centrifugation through a filter and dialyzed against 0.1 M KCl. Under this condition,

136

Figure 3. Cdc73p mutant proteins. The proposed RNA polymerase H-binding region

is indicated as a white box.

' 137

 

 

 

1" ' 554

    
 

 

interaction with
nglp/Ssu7lp or

a related function ? AP H binding

cdc73p 1-143

 

 

 

Figure 3

138

Figure 4. Production and purification of Cdc73p mutant proteins. Total E. coli

proteins were visualized 0 and 3 hr after addition of IPTG to cultures carrying production
vectors for Cdc73p, cdc73pA129-143, and cdc73p1-143. Sample sizes correspond to

0.07 A600,)“, units of bacterial cells. P indicates recombinant proteins after purification

by N12+ affinity chromatography.

139

m3;

m3d§<

.5er

 

 

 

Figure 4

140

Figure 5. The predicted interaction region of Cdc73p is important for RNA
polymerase H binding. Yeast RNA polymerase H (70 pg) was combined with Cdc73p
(20 118; lane 2), cdc73pA129-143 (20 1.1g; lane 4), or cdc73p1-143 (10 ug; lane 6) in
buffer containing 0.5 M KCl. Lane 3 contains a sample with RNA polymerase H alone
(2.5 pg). Samples were concentrated by centrifugation through an Amicon filter and
dialyzed into buffer containing 0.1 M KCl. Insoluble proteins were removed from the
solution by centrifugation, and samples were then injected into a Pharmacia Superdex
200 HR10/30 gel filtration column at a flow rate of 0.5 ml per min. RNA polymerase H
eluted at 17.5 min after sample injection. Cdc73p, cdc73pA129-143, and cdc73p1-l43
eluted at 27, 27, and 29.5 min, when fractionated in the absence of RNA polymerase H
(data not shown). Apparently, these proteins are monomeric in solution. 280 nm
absorbance peaks centered around 17.5 min, corresponding to RNA polymerase H and
bound proteins, were collected for each sample, concentrated, and analyzed by SDS-
PAGE. RNA polymerase H subunits RPB1-10 are indicated by their apparent molecular
weights in kDa along the left side of the figure. Cdc73p (lane 1), cdc73pA129-143 (lane
5), and cdc73p1-143 (lane 7) (2 ug each) are included as markers.

 

141

 

1234567

L————.———— __ __

 

Figure 5

142
binding to polymerase was expected, but some of the recombinant proteins were expected

to precipitate from solution. After removing insoluble protein by centrifugation, samples
were injected into a Superdex 200 column (Pharmacia), and the 280 nm absorbance peak
corresponding to RNA polymerase H was collected. This fraction was concentrated and
analyzed by SDS-PAGE.

Cdc7 3p bound directly to RNA polymerase H and appeared as an additional band
just above the 45 kDa RPB3 subunit (lane 2). Recombinant Cdc73p-H6 is 46 kDa, but
migrates slightly slower than expected based on its molecular weight. This protein
appears as an additional subunit of polymerase in this experiment. Similarly, the
cdc73pl-143 mutant bound to polymerase and appeared as an additional subunit (lane 6).
The cdc73pA129-143 mutant, however, was severely inhibited for polymerase binding
(lane 4). Residual interaction indicates that additional sequences to the 15 deleted amino
acids contribute to binding, but demonstrates the critival importance of this short
sequence for binding.

Cdc73p may interact with the small subunit of yeast TFHF. It is likely that
most messenger RNA genes are transcribed by the primary form of TFHF in yeast, which
includes three distinct subunits nglp/Ssu71p, ng2p, and ng3p/Anc1p. Based on the
model that Cdc73p is a component of an alternate form of TFHF, the question arises of
whether this protein cooperates with similar subunit partners to its proposed analogue,
ng2p. Cdc73p might, therefore, interact with nglp/Ssu7lp and ng3p/Anc1p, or with
alternate versions of these proteins. In Figure 6, binding between Cdc73p and
ng3p/Anc1p was tested in vitro using affinity beads on which ng3p/Anc1p was
immobilized. Cdc73p bound to these beads in buffer containing 0.1 M KCl and was at
least partially eluted with buffer containing 0.5 M KCl. Cdc73p showed no affinity for
beads with no immobilized protein ligand. This data is preliminary but may indicate a
functional interaction between Cdc73p and ng3p/Anc1p. Such an interaction would be

consistent with the proposed function for Cdc73p.

143

Figure 6. Cdc73p interacts with Anclp/ng3p. 30 111 Agarose gel beads with yeast
Anclp/ng3p (Anclp) covalently immobilized (~4 mg bound protein per ml resin) or
with no protein ligand (control) were mixed with Cdc73p (20 11g). After washing the
beads 5 times with buffer containing 0.1 M KCl, bound protein was eluted with buffer
containing 0.5 M KCl, and analyzed by SDS—PAGE developed with silver nitrate.

Lobnoo ..

322 ..,...

 

Figure 6

145
DISCUSSION

Cdc73p shares structural similarity to a TFIIF subunit and to bacterial
transcription factors sigma and delta (Figure 7). Like TFHF, sigma, and delta, Cdc73p
can be isolated based on its affinity for RNA polymerase. Cdc73p binds directly to RNA
polymerase H, and we have been able to use structural similarity to predict sequences that
are important for polymerase binding. Cdc7 3p also appears to interact with
ng3p/Anc1p, a probable transcriptional mediator that interacts with yeast TFHF and is
isolated as the smallest TFIIF subunit. Based on this preliminary analysis, we propose
that Cdc73p is a subunit of an alternate form of TFIIF in yeast. Most likely, related
functions will be discovered in other eukaryotic organisms.

The N-terminal region of RAP30 binds to the RAP74 subunit of TFIIF (11). The
N-terminal region of Cdc73p shows some similarity to RAP30 and sigma factors in this
region. Cdc73p may interact with nglp/Ssu7lp, the large subunit of yeast TFHF, or
with an alternate version of this factor that has yet to be recognized. The region 2.1
similarity appears to be a polymerase binding region of Cdc73p. C-terrrrinal regions of
Cdc73p are likely to bind to DNA, as do related structures in RAP30 and bacterial sigma
factors. This domain may be masked as are these domains in RAP30 and E. coli sigma
70 (12,13). Deletion of N-terminal sequences of Cdc73p may be important to observe
DNA binding by this factor. RAP30 also binds to TFHB (20), so Cdc73p may also bind
TFHB.

Bacteria use alternate sigma factors to recognize alternate promoter sequences to
co-regulate collections of genes (21). For examples, an alternate sigma factor in E. coli,
sigma 32, regulates heat shock genes, and multiple specific sigma factors are important in
separating developmental stages in B. subtilis sporulation. But if Cdc73p is important for
recognition of alternate promoters in eukaryotic cells, which promoters does it recognize?

RNA polymerase H transcribes several different classes of genes including messenger

146

Figure 7. Proposed similarities between yeast Cdc73p, human RAP30, and bacterial
sigma and delta factors. This figure was prepared based on sequence alignments

performed by Shi Min Fang.

147

Domains of human RAP30

TFHB binding

 

 

 

 

 

 

 

Domains of yeast Cdc73p
Function

11

 

 

 

 

Figure 7

148
RNA genes that are 3'-polyadenylated, messenger RNA genes that are not 3'-

polyadenylated (ie. histone genes in vertebrates), and small nuclear RNAs that have an
alternate 5' cap structure and are not 3'-polyadenylated. For some genes alternate 3' end
formation is determined by the promoter sequence (22), so polymerase may assemble
with a different constellation of factors at different promoters, and these factors may
cause different recognition of subsequent processing and termination signals. Therefore,
Cdc73p might be involved in transcription of a specific subset of messenger RNA genes,
ie. histone genes, or small nuclear RNA genes. If Cdc73p were required for transcription
of histone genes, for instance, this might provide clues as to the mechanism of cell cycle
arrest in cdc73 mutants, since histone synthesis is required for the GW transition.
Alternatively, Cdc7 3p might be a transcriptional repressor that functions by antagonizing
the function of ng2p through competition for polymerase binding.

Since temperature sensitive cdc73 mutants are available (2), RNA levels produced
from different genes can be monitored under permissive and non-permissive conditions.
Such an approach may permit the identification of genes controlled by Cdc73p. Two
hybrid interaction screens may be useful to identify proteins that bind to Cdc73p, for
instance to determine whether nglp/Ssu7 1p or another protein binds to the N-terrninal
region, as predicted by our model. Affinity tagging Cdc73p in vivo is another approach
to identifying additional transcription factors that interact. Using a combination of in
vivo and in vitro approaches, therefore, the function of Cdc73p in transcription should be
revealed by these studies.

Since TFHF plays a central function in transcription, an alternate TFHF might be
expected to support a substantially different mechanism. For instance, TFIIE and TFIH-I
might be dispensible for a Cdc73p dependent initiation mechanism. A mechanism that
did not involve TFHH would likely not require ATP 13—7 bond hydrolysis for initiation,
since this is a presumed function of the TFIIH helicase activity (23). If TFIHT were not
part of the mechanism, the CTD would not be phosphorylated by the CTD kinase that is

149
another component of TFIH-I (24). CID phosphorylation is thought to regulate promoter

entry (25), promoter escape (26, 27), and elongation (28); therefore, a mechanism that did
not involve phosphorylation might have less processive transcription and an alternate 3'
end formation. For instance, small nuclear RNAs are much shorter transcripts than most
mRNA transcripts, so their synthesis would not require as processive a polymerase. A
dephosphorylated CTD might associate with an alternate set of processing and
termination factors.

Interestingly, some small nuclear RNAs are synthesized by RNA polymerase H
and some are synthesized by RNA polymerase IH (29). Promoters that are recognized by
one or the other polymerase are very similar and both polymerases utilize the same TBP-
TAF complex to transcribe these genes (30). Other factor and co-factor requirements for
transcription of these genes are less clearly defined. Whether T'FIIB, TFIIF, TFIIE,
TFHH, or ATP hydrolysis are required has not been reported. A few straightforward
experiments using existing in vitro systems might indicate the possibility of an alternate

TFIIF for transcription of these genes.

ACKNOWLEDGEMENT

We thank laboratory members S.-M. Fang for computer analysis, S. Reymez for
yeast RNA polymerase H, and L. Lei for immobilized Anclp. This research was
supported by the National Institutes of Health, Michigan State University, The Michigan
State University Agricultural Experiment Station.

150
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