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THESIS

 

 

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293 02074 1173
Mlchlgan State
University

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This is to certify that the

dissertation entitled

INTERACTIONS OF TFIIF IN THE PREINITIATION COMPLEX

presented by

STEPHAN REIMERS
has been accepted towards fulfillment

of the requirements fer

Ms. degree in BIOCHEMISTRY

A4313. \B <1;

ajorfi ro essor

Date [451”!99 / ‘/

l

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
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11/00 WWI-.9659.“

INTERACTIONS OF TFIIF IN THE PREINITIATION COMPLEX
By

Stephan Reimers

A THESIS

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

MASTER OF SCIENCE

Department Of Biochemistry

1999

ABSTRACT

INTERACTIONS OF TFIIF IN THE PRETNITIATION COMPLEX

By

Stephan Reimers

Transcription factor TFIIB is an essential component of the RNA polymerase II
initiation complex. TFIIB acts as a bridging factor by stabilizing the TBP-TATA box
interaction on the promoter and by recruiting RNA polymerase II and TFIIF. Several
lines of genetic and in vitro experiments implicate that transcriptional start site selection
is mediated by a complex that involves TFIIF, TFIIB, and polymerase. The N-terminus of
TFIIB has long been postulated to be a scaffold for the assembly Of RNA polymerase II-
TFIIF. The N-terminal domain Of TFIIB binds the RAP3O subunit of TFIIF and RNA
polymerase II. RAP74, the large subunit Of TFIIF, has been shown to interact genetically
with the N-terminus of TFIIB. In addition to the functional interaction between TFIIB
and TFIIF during transcription initiation, both factors may cooperate to regulate
dephosphorylation of the Carboxy Terminal Domain (CTD) of RNA polymerase 11 after
transcription termination. Here, we characterize the interaction between TFIIF and TFIIB
using surface plasmon resonance (SPR) / BIACORE. We demonstrate that TFIIF binds
the N-terminal domain Of TFIIB. Our studies also indicate that the interaction Of the
TFIIF complex with TFIIB occurs largely through the C-terminus of RAP74. A
truncation Of the last 25 amino acids of RAP74 abrogates the binding Of TFIIF with

TFIIB. Using TFIIF amino acid substitution mutants, we further characterize the region

Of RAP74 between 475 and 505 as being important for TFIIB interaction. Through gel
mobility shift assays, we also show that the N-terminus of TFIIB is not required for the
recruitment of RNA polymerase II and TFIIF in vitro. TFIIB sequence from amino acid
11 to 103, encompassing the zinc finger domain, is dispensable for the assembly Of
DBPOlF complexes. However, amino acid sequence between 11 to 45 is required for

basal transcription.

This is dedicated to my wife and family,
whose love and support have kept me grounded
to what is important in life.

iv

ACKNOWLEDGEMENTS

I would like to thank my mentor, Dr. Zach Burton, whose knowledge, support,
efforts, patience, and accomplishments have served as an inspiration toward my
development as a scientist. I extend a warm hand of gratitude tO Drs. Ann Finkelstein, Lei
Lei, Delin Ren, and David McConnell for their friendship and scientific expertise

throughout the years.

TABLE OF CONTENTS

Page
LIST OF TABLES .......................................................................... viii
LIST OF FIGURES ......................................................................... ix
LIST OF ABBREVIATIONS ............................................................. xi
CHAPTER 1
LITERATURE REVIEW ................................................................ 1
Overview ..................................................................................... 2
T RANSCRIPT IONAL MA CHINERY ...................................................... 5
Promoter Structure .......................................................................... 5
RNA Polymerase II (RNAPII) ............................................................ 7
Carboxy Terminal Domain (CTD) .............................................. 7
Holoenzymes ....................................................................... 10
Basal Transcription Factors ............................................................... 11
Transcription Factor IID (TFIID) ................................................ 11
TATA Box Binding Protein (TBP) .............................................. 12
TBP-Associating Factors (TAFns) ............................................... 14
Transcription Factor IIA (TFIIA) ................................................ l7
Transcription Factor IIB (TFIIB) ................................................ 18
Transcription Factor IIF (TFIIF) ................................................. 19
Transcription Factor IIE (TFIIE) ................................................. 26
Transcription Factor IIH (TFIIH) ................................................ 27
TRANSCRIPTION BY RNA POL YMERASE II .......................................... 29
Initiation ............................................................................. 29
Preinitiation Complex Assembly (PIC) .......................................... 29
Open Complex Formation ......................................................... 34
Abortive Initiation .................................................................. 35
Promoter Escape/Clearance ........................................................ 35
Elongation ............................................................................ 37
Pausing, Arrest, and Termination ................................................. 38
Elongation Factors .................................................................. 39
Termination and Recycling ........................................................ 42

vi

TRANSCRIPTIONAL REG ULA TION ..................................................... 44

Transcriptional Activation .................................................................. 44
Activators ............................................................................ 45
Coactivators ......................................................................... 46
Template Activation ................................................................ 47
Recruitment Of GTFs ............................................................... 48
Stimulation of Promoter Escape and Elongation ............................... 51

CHAPTER 2

INTERACTION BETWEEN TRANSCRIPTION

FACTORS TFIIF AND TFIIB .......................................................... 54

Introduction .................................................................................. 55

Materials and Methods ..................................................................... 61

Results ........................................................................................ 67

Discussion .................................................................................... 122

REFERENCES .............................................................................. 132

vii

LIST OF TABLES

Page
1. Dissociation constants (KB) of RAP30, RAP74, TFIIF with TFIIB ............. 81

2. Summary Of TFIIF point mutants transcription and interaction with TFIIB. . .. 95

viii

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 14.

Figure 15.

LIST OF FIGURES

Page
N- and C-terminal domains of RAP74 ................................. 60
SDS-PAGE of TFIIF C-terminal truncation protein
complexes stained with Coomassie blue ................................ 69
Single and Multiple Round Transcription with TFIIF
C-terminal truncations 1-217, 1-492, 1- 472 ............................ 72
Binding kinetics analysis of TFIIF subunits RAP3O
and RAP74 binding immobilized TFIIB ................................ 75
Binding kinetics analysis of wild type TFIIF binding
immobilized TFIIB protein ................................................ 77
Structural and functional domains Of TFIIB and SDS-PAGE
Of TFIIB truncation mutants stained with Coomassie blue ........... 79
Kinetic analysis Of TFIIF binding immobilized TFIIB-N (1- 113)
and TFIIB- C (114- 316) .................................................... 83
Qualitative BIACORE analysis of RAP74 and TFIIF truncation
mutants complexes binding to immobilized TFIIB .................... 85
Alignment Of human, Drosophila, and S. cerevisiae of the
C-terminal region Of RAP74 .............................................. 88
Single and Multiple Round Transcription with TFIIF C-terminal
point mutants ................................................................. 91
TFIIF C-terminal deletion and point mutants in a purified
transcription assay system ................................................... 94

BIACORE qualitative analysis Of TFIIF point mutation complexes
capable of binding to immobilized TFIIB ................................ 97

BIACORE qualitative analysis Of TFIIF point mutation complexes
compromised for TFIIB binding ........................................... 99

Summary Of mutant TFIIB proteins obtained from M. Horikoshi.... 102

SDS-PAGE Of TFIIB mutants stained with Coomassie blue.. . . . . . 104

ix

Figure 16.

Figure 17.

Figure 18.

Figure 19.

Figure 20.

Figure 21.

Page

Gel mobility shift assay of TFIIB N-tenninal, C-terminal
deletion mutants ............................................................. 109

Gel mobility shift assay of TFIIB internal deletion mutants. . . . . . 111

Gel mobility shift assay of TFIIB N-terminal, C-terminal deletion
mutants using TFIIF (1-217) ............................................... 113

Gel mobility shift assay Of TFIIB internal deletion mutants using
TFIIF (1-217) ............................................................... 115

Compensation of the defective TFIIB internal deletion A208-227
with TFIIE .................................................................... 117

TFIIB N- and C-terminal mutants in a purified transcription assay
system ......................................................................... 121

a.a.
AdMLP
ATP
BRE
CTD
CTP
DNA
GTP
HAT
HCA
Inr

kDa
mRN A
NTP
PAGE
PCR
RAP
RN APII
sarkosyl
SDS
TFII

UTP

LIST OF ABBREVIATIONS

amino acid(s)

adenovirus major late promoter
adenosine triphosphate

TFIIB recognition element

carboxyl terminal domain Of the largest subunit of RNA polymerase II
cytidine triphosphate

deoxyribonucleic acid

guanosine triphosphate

histone acetyltransferase

hydrophobic cluster analysis

Initiator element

kilodaltons

messenger RNA

nucleoside triphosphate
polyacrylamide gel electrophoresis
polymerase chain reaction

RNA polymerase II associating protein
RNA polymerase II
N-lauroylsarcosine sodium salt

sodium dodecyl sulfate

transcription factor Of RNA polymerase II

Uridine triphosphate

xi

CHAPTER 1

LITERATURE REVIEW

Overview

The regulation Of gene expression is at the core Of cellular function and
metabolism. RNA polymerase 11 requires the complex interaction of transcription factors
with the basal RNA polymerase II transcription machinery to form the functional
holoenzyme responsible for the transcription Of protein-coding genes. RNA polymerase
II underlies development and differentiation, it is the endpoint Of signal transduction
pathways and it reshapes the cell in response to metabolic need and specific
environmental information. Understanding the mechanistic details by which these
processes occur has a direct impact on medical problems, such as some forms of cancer
in which there is a strong correlation between loss of growth inhibition and aberrant
transcriptional regulation.

Control of transcription in eukaryotes requires a greater repertoire of accessory
proteins than it does in prokaryotes. In prokaryotes, a single core RNA polymerase
enzyme composed of three subunits is responsible for all cellular transcription. Bacterial
RNA polymerase consists of an OLZBB’ core enzyme and a 0' factor to confer promoter
recognition and specificity (Platt, 1986). Eukaryotic cells contain three nuclear DNA-
dependent RNA polymerases that transcribe different sets of genes. RNA polymerase I
(A) synthesizes ribosomal RNA precursors. RNA polymerase II (B) transcribes pre-
messenger RNA (mRN A) and some small nuclear RNA (snRN A). RNA polymerase III
(C) transcribes SS rRNA and transfer RNA (tRNA) and some snRNA. Each Of these
enzymes requires the aid of an assortment of additional factors for selective promoter

recognition and regulated transcription initiation.

Eukaryotic mRN A synthesis proceeds through multiple stages referred to as
preinitiation, initiation, elongation, and termination. As mentioned earlier, RNAP II alone
is not sufficient for specific initiation (Roeder, 1976; Weil et al., 1979). Polymerase
requires additional sets of protein factors for accurate initiation from promoter DNA in
vitro (Matsui et al., 1980). These accessory factors are termed GTFs (general
transcription factors) and include TFIID, TFIIA, TFIIB, TFIIF, TFIIE and TFIIH. These
GTFs function in intimate association with RNA polymerase II and are required for
selective binding of polymerase to its promoters, formation of the open complex, and
synthesis of the first few phosphodiester bonds of nascent transcripts.

The conventional model for ordered transcription initiation by RNAPII is
characterized by a distinct series of events: 1) recognition Of the promoter DNA by
TFIID, 2) recognition of the TFIIB-promoter complex by TFIIB, 3) recruitment of a
TFIIF/RNAP II complex, 4) binding of TFIIE and TFIIH to complete the preinitiation
complex, 5) promoter melting and formation Of an “Open” initiation complex, 6)
synthesis of the first phosphodiester bond Of the nascent mRNA transcript, 7) release Of
RNAP 11 contacts with the promoter (escape), and 8) elongation Of the RNA transcript.
RNAP II and the GTFs can assemble in a defined order on promoter DNA in vitro,
suggesting stepwise assembly Of the preinitiation complex (Buratowski et al., 1989; Van
Dyke et al., 1988). However, similar to the initiation Of transcription by bacterial
polymerase holoenzyme (Busby and Ebright, 1994), in vivo RNAP 11 may assemble into
a multi-component holoenzyme complex before binding to promoter DNA. RNA
polymerase II holoenzymes have been purified from many eukaryotic organisms (Chao et

al., 1996; Cho etal., 1997; Kim et al., 1994; Koleske and Young, 1994; Maldonado et al.,

1996; Ossipow et al., 1995; Pan et al., 1997; Thompson et al., 1993). The subunit
composition of these different preparations differs somewhat, and these differences
involve the presence or absence Of GTFs and regulatory factors. The existence of this
RN APII holoenzyme suggests an alternative to the paradigm of sequential assembly of
GTFs and suggests that most of the initiation machinery can bind to a promoter in a
single step.

Sequence-specific DNA binding proteins regulate mRNA transcription in gene-
specific ways (Mitchell and Tjian, 1989). Activated transcription involves the basal
transcription apparatus integrating with additional factors (activators/repressors) that bind
elements upstream Of the promoter DNA. Most transcriptional regulation in eukaryotes is
believed to be mediated directly by transactivators or through the intercession of adapters
to modulate the rate of initiation and/or elongation by RNAP II.

Transcription factors cover a wide range of functions in the regulation of accurate
transcription. General transcription factors have roles in pre-initiation, initiation, and
elongation. This chapter will review the basics Of mRNA transcription by RNAPII on the
canonical Adenovirus Major Late Promoter (AdMLP) and will include a treatise of the
components of the general transcriptional machinery and the process of basal
transcription. A summary Of transcriptional regulation including activation and

repression will also be given.

TRANSCRIPTIONAL MACHINERY

Promoter Structure

Three distinct families Of DNA sequence elements direct transcription by RNAP
II. The first family includes the core/basal promoter elements found near the site where
RN AP 11 initiates transcription. Two classes of core elements have been identified: the
TATA element, located 25-30 base pairs upstream of the transcription start site with a
consensus sequence TATA(A/T)A(A/T) and the Initiator (Inr) motif, a pyrimidine-rich
sequence PyPyA+.N(T/A)PyPy encompassing the transcription start site (Weis and
Reinberg, 1992). The TATA and Inr core elements serve to nucleate the initiation
complex and are recognized by components of the transcription machinery. They can
function independently or synergistically. Various promoters contain one element or both
elements. Two other families Of cis-regulatory elements are the promoter-proximal
elements like CCAAT and GC elements, situated 50 to a few hundred base pairs
upstream Of the start site, and the promoter-distal elements (enhancers) found up to tens
of thousands of base pairs away from the transcription start site (Mitchell and Tjian,
1989; Ptashne, '1 989). These elements contain binding sites for protein that modulate
transcription through activation or repression. Transcriptional activators bind to these
elements and facilitate assembly of the preinitiation complex through direct or indirect
contact with the general transcription factors. The binding of transcriptional repressors to
these elements impair transcription by interfering with activators or by inhibiting general
transcription factors. It is the combination of all these elements that give gene-specific

promoters their characteristic strength.

A new class Of core promoter element located downstream Of the Inr has been
identified in Drosophila. The downstream promoter element (DPE) with a conserved
sequence motif (A/G)G(A/T)CGTG, is located about 30 bp downstream of the start site
(Burke and Kadonaga, 1996). It is a basal promoter element that is present in a subset of
Drosophila TATA-box-deficient (TATA-less). DNase I footprinting and in vitro
transcription experiments revealed that a DPE in its normal downstream location is
necessary for transcription of DPE-containing TATA-less promoters and can compensate
for the disruption of an upstream TATA box of a TATA-containing promoter (Burke and
Kadonaga, 1997). The DPE works in conjunction with the Inr element to provide a
binding site for TFIID in the absence Of a TATA box to mediate transcription of TATA-
less promoters. Two components of TFIID, Drosophila TAFn60 and TAFn 40 have been
found to interact specifically with the DPE (Burke and Kadonaga, 1997).

In addition to the TATA, the initiator, and the downstream promoter element, a
fourth core promoter element has recently been identified (Lagrange et al., 1998). The
TFII_I_3_ recognition element (BRE) is located immediately upstream of the TATA element
and has a consensus sequence of (G/C)(G/C)(G/A)CGCC. As the name indicates, the
BRE is specifically recognized by the general transcription factor TFIIB. It is possible
that TFIIB-BRE interaction may play a role in determining the overall strength of a
promoter, the order of preinitiation complex assembly at a promoter, the rate-limiting step
in transcription initiation at a promoter, and the responsiveness of a promoter to specific

transcriptional activators.

RNA Polymerase 11 (RN APII)

RNA Polymerase II is a highly conserved multisubunit protein complex among
eukaryotes (Young, 1991).The three largest subunits, prl, pr2 and pr3, are related
to the prokaryotic core RNA polymerase subunits and are largely responsible for RNA
catalysis (Markovtsov et al., 1996; Mustaev et al., 1997; Severinov et al., 1996;
Zaychikov et al., 1996). RNAPII is generally composed of 10 to 12 subunits. Hela cell
RNAPII contains 10 subunits ranging in size from 240 kDa to 10 kDa. Yeast RNAPII
consists Of 12 polypeptides with molecular sizes of 220 kDa to 10 kDa. The two largest
subunits RPBl and RPB2 are structurally and functionally related to the two largest
subunits Of E. coli RNA polymerase 13’ and B, respectively (Sweetser et al., 1987). Both
subunits are involved in DNA and nucleotide substrate binding. The two large subunits Of
yeast RNA polymerase I and III are also homologues Of E. coli RNA polymerase [3’ and
[3 subunits. pr3, the third largest subunit, and pr11 are related to the or subunit of
E. coli RNA polymerase (KOlOdziej and Young, 1991) and may play a role in nucleating
assembly of the enzyme. Five smaller subunits (prS, pr6, and pr8, pr10, and
pr12) Of 14-28 kDa are essential components shared by all three nuclear RNA
polymerases in eukaryotes (Allison et al., 1985; Memet et al., 1988; Treich et al., 1992;

Woychik et al., 1990).

Carboxy Terminal Domain (CTD)
The largest subunit of eukaryotic RNAPII (pr1) contains an unusual C-terrninal
domain (CTD) consisting of multiple repeats of the consensus sequence Tyr-Ser-PrO-Thr-

Ser-PrO-Ser (YSPTSPS) (Corden et al., 1985). This domain is not present in eukaryotic

RNAPI, RNAPIII, and prokaryotic RNA polymerases. The number of repeats of the
heptapeptide consensus sequence appears to correlate with the genetic complexity of the
organism. The sequence is repeated with some degeneracy 26-27 times in yeast, 42-44
times in Drosophila, and 52 times in mammals (Sawadogo and Sentenac, 1990; Young,
1991). Due tO its high serine, the CTD can be highly phosphorylated at the Ser-Pro
repeats. As a result of phosphorylation, RPBl is frequently resolved into two major
forms, the largely unphosphorylated Ila form or the highly phosphorylated 110 form
(Cadena and Dahmus, 1987; Kim and Dahmus, 1988). A third form that is sometimes
isolated lacks the CTD (designated as RNAPIIb) as a result of truncation due to
proteolysis. Deletion mutations that remove the CTD are lethal in yeast (N onet et al.,
1987) , Drosophila (Zehring et al., 1988) and mouse (Bartolomei et al., 1988). Partial
truncation Of the CTD in yeast has been shown to confer cold-sensitivity and inositol
auxotrophy (Nonet and Young, 1989). One possible function of the CTD may be to
mediate transcription activation by upstream regulators. Except with Spl (Zehring and
Greenleaf, 1990) , the effects Of activator proteins are abolished when CTD truncation
mutants are introduced in RN APII Of mammalian cells (Gerber et al., 1995).

The CTD becomes extensively phosphorylated during the initiation of
transcription and must be dephosphorylated for re-entry of the polymerase in an initiation
complex (Lu et al., 1991; O. Brien et al., 1994). The phosphorylation is thought to play a
role in the transition from initiation to elongation (Cadena and Dahmus, 1987; O. Brien et
al., 1994). Multiple kinases appear to mediate CTD. phosphorylation. Most notably, the
kinase activity Of TFIIH (Feaver et al., 1991; Lu et al., 1992; Serizawa et al., 1992) and

P-TEFb (positive transcription elongation factor b) (Marshall et al., 1996) can

phosphorylate the CTD. The SrblO/Srbll kinase cyclin pair (Liao et al., 1995), Cdc2 , a
homologous protein to P-TEFb’s PITARLE catalytic subunit (Cisek and Corden, 1989),
and Ctkl (Lee and Greenleaf, 1991) also have kinase activities that can phosphorylate the
CTD. Whether these CTD kinases are gene specific or affect different steps during the
transcription cycle remains to be determined. Recently, extensive ubiquitination Of pr1
during transcription reactions in vitro was Observed (Mitsui and Sharp, 1999).
Phosphorylation of the CTD was a signal for ubiquitination. Specific inhibitors Of the
CTD cyclin-dependent kinase (cdk) complex suppressed the ubiquitination reaction.
Another study demonstrated that UV-induced ubiquitination Of phosphorylated CTD
prl was followed by its degradation in the proteasome (Ratner et al., 1998).
Interestingly, a proteasomal subunit (SUGl) copurifies with a CTD kinase / DNA repair
complex, TFIIH (Friedberg, 1996; Weeda et al., 1997). This presents a model that in both
regulation of transcription and DNA repair, phosphorylation Of the CTD by kinases might
signal degradation Of the polymerase. Transcriptional arrest Of RNAPII at DNA lesions
could be aborted through proteasome degradation rather than through resumption of
elongation after DNA repair.

CTD kinases and CTD phosphatases may contribute to mediating differential
CTD phosphorylation. A phosphatase activity specific for dephosphorylation Of the CTD
has been identified (Archambault et al., 1997; Chambers and Dahmus, 1994; Chambers
and Kane, 1996; Cho et al., 1999; Dubois et al., 1999). This CTD phosphatase activity is
regulated by both TFIIF and TFIIB (Chambers et al., 1995). The RAP74 subunit of
TFIIF stimulates CTD phosphatase activity while TFIIB inhibits this stimulatory effect Of

TFIIF. The CTD phosphatase allows recycling of RNAPII because the

hypophosphorylated IIA form of polymerase preferentially enters the preinitiation
complex. The phosphatase dephosphorylates the CTD allowing efficient incorporation of

RNAPII into transcription initiation complexes, which results in increased transcription.

Holoenzymes

RNA polymerase 11 requires multiple general transcription factors to initiate site-
specific transcription. These proteins have been shown to assemble in vitro in an ordered
fashion onto promoter DNA (Buratowski etal., 1989; Van Dyke et al., 1988) .Recent
evidence indicates that many of the polymerase’s accessory factors assemble into a large
complex, called the RNA polymerase holoenzyme. Holoenzyme is a form Of RNA
polymerase II that potentially is readily recruited to promoters in vivo. Characterization
of the yeast holoenzyme has identified several initiation factors including TFIIB, TFIIF,
TFIIH, SRB/mediator complex and other regulatory proteins (Kim et al., 1994; Koleske
and Young, 1994). Several forms of mammalian RNA polymerase II holoenzymes have
also been isolated (Chao et al., 1996; Maldonado et al., 1996; Ossipow et al., 1995; Pan et
al., 1997; Scully et al., 1997). Unlike core RNA polymerase 11, holoenzyme responds to
transcriptional activators in vitro. Transcription by RNAPII holoenzyme is stimulated by
the activator protein GAL4-VP16 (Koleske and Young, 1994). Other distinct forms Of
yeast RNAP 11 holoenzyme have been discovered containing various subsets of general
initiation factors (Kim et al., 1994; Koleske and Young, 1994), the SWI/SNF chromatin
remodeling complex (Wilson et al., 1996), and Pafl /Cdc73 complexes that regulate only
a subset of genes (Shi et al., 1997; Shi et al., 1996; Wade et al., 1996). These

holoenzymes differ in their composition but all respond to transcriptional activators.

10

Basal Transcription Factors

The initiation of mRN A transcription is an essential stage in the regulation of
gene expression. For RNA polymerase II to transcribe a gene, an array Of more than 20
polypeptides must assemble at the promoter. A universal set Of proteins, termed the basal
apparatus, recognizes a core promoter and initiates transcription. This basal apparatus
comprises polymerase II and the general transcription factors (GTFs) TFIID (TBP +
TAFs), TFIIB, TFIIA, TFIIE, TFIIH, and TFIIF. Almost all core promoters employ the
same set of basal transcription factors and the subunits are conserved between yeast and

man.

TFIID

Transcription factor TFIID is a multisubunit protein complex (z 750 kDa)
essential for RNAPII transcription from class II promoters. It was initially isolated from a
Drosophila nuclear extract as a complex capable of binding the TATA DNA sequence
(Parker and Topol, 1984). Mammalian TFIID was identified as a chromatographic
fraction of Hela cell nuclear extract, capable of protecting the TATA box and regions
therein on the Adenovirus Major Late Promoter (AdMLP) (Horikoshi et al., 1988;
Reinberg et al., 1987). The TFIID complex consists of the TATA binding protein (TBP)
and several accessory proteins called IBP- associated factors (TAFus) (Burley and
Roeder, 1996; Gill and Tjian, 1992; Goodrich and Tjian, 1994; Hernandez, 1993).
TFIID’s promoter binding activity makes it one of the most important molecules in
RNAPII transcription. It is responsible for nucleating the assembly of a transcriptionally

competent initiation complex (Nakajima etal., 1988; Zawel and Reinberg, 1993).

ll

Recognition of the core promoter by TFIID is likely to be one of the first rate-limiting
steps in the transcription initiation process (Buratowski et al., 1989). Two other TBP-
TAF complexes exist in the nucleus of eukaryotic cells for transcription by RNAPI and
RNAPIII. Distinct sets Of TAFus in these TBP-TAF complexes provide promoter
selectivity for transcription by these polymerases. SL1 is the TBP-TAF required for
RNAPI transcription of ribosomal RNA (Comai et al., 1992). RNAPIII transcription of
transfer RNA, SS RNA and small nuclear RNA is carried out through TBP-TAF
complexes, designated TFIIIB (Huet and Sentenac, 1992; Kassavetis etal., 1992; Lobo et
al., 1992; Simmen et al., 1992; Taggart et al., 1992) and SNAPc (Hernandez, 1993).
SNAPc is a bifunctional complex that, depending on snRNA structure, directs either

RNAP III or RNAP II transcription Of snRNA (Hernandez, 1993).

TBP

TATA Binding Protein’s (TBP) primary function is promoter recognition through
binding of the TATA box. TBP is an essential transcription factor for accurate
transcription and plays a requisite role in transcription initiation by all three RNA
polymerases (Cormack and Struhl, 1992; Sharp, 1992). TBP is also important for
nucleation of functional preinitiation complexes on TATA-less promoters (Martinez et
al., 1994; Weis and Reinberg, 1992). Amino acid sequence analysis of TBP from yeast,
Drosophila, Arabidopsis and human reveals that the C-terminal domain of the protein
comprising 180 amino acids is highly conserved (Hernandez, 1993; Pugh and Tjian,
1990). Basal in vitro transcription experiments have shown yeast TBP to be functionally

interchangeable with mammalian TBP (Cavallini et al., 1988; Hahn et al., 1989).

12

However, human TBP fails to functionally substitute yeast TBP in vivo (Cormack et al.,
1991; Gill and Tjian, 1991). Although highly conserved and functionally analogous,
TBP’s molecular weight ranges from 22 kDa (Arabidopsis), 27kDa (yeast), to 38 kDa
(human and Drosophila) (Burley and Roeder, 1996).

The conserved C-terminal domain of TBP or as it is called TBP “core domain”
(TBPc) forms the basis of two imperfect direct repeats that are sufficient for both binding
to the TATA box and basal transcription (Hoey et al., 1990; Horikoshi et al., 1990;
Peterson et al., 1990) . This domain is also important for interaction with general
transcription factors TFIIA and TFIIB (Buratowski et al., 1989; Maldonado et al., 1990).
TBPc has also been shown to be sufficient for support of normal cellular growth in yeast
in vivo (Lieberman et al., 1991).

Crystal structures ofArabidopsis, yeast and human TBPc and TBPc bound to
DNA have been solved (Chasman et al., 1993; Kim et al., 1993; Kim et al., 1993;
Nikolov et al., 1996; Nikolov et al., 1992). The structure Of the molecule resembles a
“saddle” made up Of two equal pseudo-symmetrical halves. Each Ot/B domain
corresponds to each of the C-terminal direct repeats, composing two or helices and five
antiparallel B—sheets that are connected in the order Sl-Hl-S2-S3-S4-SS-H2 (S for [5-
sheet; H for Ot-helix). The Ot-helical loop between the first and second B-sheets (SI-H1-
82) form the basis of the “stirrups” represented on both sides of the molecule that are
responsible for interactions with TFIIA and TFIIB (Buratowski et al., 1989; Maldonado
et al., 1990). TBP sits astride the DNA and recognizes its binding site through minor
groove contacts (Lee et al., 1991; Starr and Hawley, 1991). Binding of TBP to the TATA

box induces extensive distortion in the DNA helix and introduces a severe 80° kink or

13

bend in the DNA (Horikoshi et al., 1992). This bending serves to increase the proximity
of other factors forming the preinitiation complex and may also serve to align upstream
regulatory factors closer to the basal machinery.

The N-terminal domain of TBP is less well-conserved in eukaryotes (Cormack et
al., 1991; Gill and Tjian, 1991) and may mediate interactions with coactivators (Pugh and
Tjian, 1990; Zhou et al., 1991). However, the N-terminus among vertebrate forms of TBP

is well conserved and regulates RNA polymerase III transcription at the U6 promoter

(Mittal and Hernandez, 1997).

TAFus

Although TBP is sufficient for promoter recognition and the assembly Of
subsequent general transcription factors into a functional preinitiation complex,
transcriptional activation is observed only when TFIID is used (Hoffman et al., 1990;
Pugh and Tjian, 1990). TBP is the universal transcription factor that is required by all
three RNA polymerases (Hernandez, 1993). The presence of distinct TBP Associating
Factors (TAFus) bound to TBP programs these transcription complexes to discriminate
between promoters designated for different classes of RNA polymerases. In each case, it
is decorated with distinct sets of associated factors. Drosophila TFIID has been entirely
reconstituted from recombinant proteins of TBP and eight TAFns with molecular weight
of 30 to 250 kDa (Chen et al., 1994). Human TFIID, contains 8 to 11 additional TAFus
ranging from 15 to 250 kDa to form the complex (Burley and Roeder, 1996; Goodrich
and Tjian, 1994; Hampsey and Reinberg, 1997). Mammalian homologues have been

described for all the Drosophila TAFns. In yeast, TFIID lacks an evident homologue of

14

dTAF [11 10 /hTAF 11130 and hTAF "105 (Dikstein et al., 1996). The largest Drosophila
TAF, dTAF11250 and its human and yeast counterpart are critical in the assembly of
TFIID and help anchor TBP in the TFIID complex (Chen et al., 1994). TAFus appear to
play multiple roles in transcription that include promoter recognition, catalysis, and can
serve as activation domain targets. TAFns are important in recognizing core promoter
structures including both the Inr and DPE (Burke and Kadonaga, 1997; Hansen and
Tjian, 1995; Kaufrnann and Smale, 1994; Purnell et al., 1994; Verrijzer et al., 1995). The
different sequence recognition properties of TBP and TAFus may allow TFIID to
recognize different promoter elements, either individually or in combination, thereby
permitting activation from a diverse array of promoters (Roeder, 1996). In addition to
participating in functionally relevant interactions with promoter DNA, TAFns contact
other GTFs in the preinitiation complex (Goodrich et al., 1993; Hisatake et al., 1995;
Ruppert and Tjian, 1995; Yokomori et al., 1993).

It was observed that dTAFn30, dTAF 1140, dTAF 1160 and their human homologues
are related in sequence and structure to the core histone proteins H2B, H3, and H4
(Burley and Roeder, 1996). Biochemical and crystallographic analyses indicate that a
histone octamer-like structure may exist within TFIID (Hoffrnann et al., 1996; Hoffinann
et al., 1997). This raises the possibility that TAFus bind DNA in a similar fashion to the
their core histone counterparts. Mimicry of a nucleosome like structure changes the
topology of the promoter and could allow wrapping of DNA to permit the multiple
contacts by TFIID with core promoter elements or with transcriptional activators.

TFIID possesses two known catalytic activities, including histone acetylation and

basal factor phosphorylation. TFIID’s histone acetyl-transferase (HAT) activity resides in

15

hTAFu250 (Mizzen et al., 1996). The HAT activity of TAF11250 is conserved in yeast,
Drosophila, and man. Histone acetylation is an important step in the conversion of
inactive chromatin into a transcriptionally competent form. Acetylation of lysine residues
in histones weakens histone-DNA interactions, thereby permitting access to the DNA by
activators and general transcription factors. TAFn250 is a bipartite protein kinase which
can phosphorylate itself and the RAP74 subunit of TFIIF (Dikstein et al., 1996).
Sequence alignment between human TAF11250 and its yeast counterpart TAF “145/
TAFul30 reveal that neither the kinase domain nor the TFIIF interaction domain is
conserved in the yeast protein. Phosphorylation of TFIIF in higher eukaryotes may
influence activator-dependent recruitment of RNA polymerase 11 into promoter
complexes or may affect the initiation and elongation properties Of RNA polymerase II.
TAFns can serve as coactivators to receive gene-specific transcriptional
activation signals. TFIID is the target of many activator domains as the binding Of
activators to TAFus can enhance recruitment of TFIID to the promoter. TBP can replace
TFIID fractions in basal transcription, but fails to support activated transcription in
human and Drosophila systems in vitro (Chen et al., 1994; Chiang and Roeder, 1995;
Dynlacht et al., 1991; Jacq et al., 1994; Pugh and Tjian, 1990; Tanese et al., 1991). TFIID
fractions containing TAFns are essential for regulated transcription. As such, numerous
activator-TAP interactions have been described (Pugh, 1996; Zawel and Reinberg, 1995).
Gln-rich Spl and Bicoid bind dTAFul 10 (Gill et al., 1994; Sauer et al., 1995; Sauer et al.,
1995). Acidic activation domain proteins VP16 and p53 bind dTAFn40 and dTAFu60

(Goodrich et al., 1993; Klemm et al., 1995).

16

TFIIA

Yeast TFIIA is a heterodimer Of 32 kDa and 13.5 kDa (Ranish and Hahn, 1991).
Deletion of either of the yeast genes encoding the two polypeptides (TOAl and TOA2)
is lethal (Ranish et al., 1992). Human TFIIA (DeJong et al., 1995; DeJong and Roeder,
1993; Ma et al., 1993; Ozer et al., 1994; Sun et al., 1994) and Drosophila TFIIA
(Yokomori et al., 1993; Yokomori et al., 1994) are composed of three subunits with
homology to the yeast proteins. The 37 kDa a and 19 kDa [3 subunits share sequence
similarity with the large subunit in yeast, while the 13 kDa y subunit is homologous to
the smallest yeast subunit. The on and [3 subunits of human and Drosophila TFIIA are
generated by protein processing from a single polypeptide precursor.

TFIIA facilitates and stabilizes the binding of TFIID to DNA through direct
interaction with TBP (Buratowski and Zhou, 1992; Imbalzano et al., 1994; Lee et al.,
1992). X-ray crystallographic structures of TBP-DNA-TFIIA complexes have been
solved (Geiger et al., 1996; Tan et al., 1996). TFIIA binds to the N-terminal stirrup
structure of TBP and makes contact with the DNA backbone upstream of the TATA box.
TFIIA binds with high affinity to both TBP and certain TAFns (Yokomori et al., 1993) in
the TFIID complex. It has been shown that cloned TFIIA plays no apparent role in basal
transcription and is in fact dispensable for accurate initiation in in vitro transcription
systems when TBP and not TFIID is used (Sayre et al., 1992; Sun et al., 1994; Yokomori
et al., 1994). However, TFIIA plays an essential role in in viva transcription, because of
the lethal phenotype it displays when its yeast genes are deleted (Ranish et al., 1992).

Despite TFIIA’s ambiguous role in in vitro basal transcription, TFIIA is required

for activator stimulated transcription in vitro (Ma et al., 1993; Ozer et al., 1994; Sun et

17

al., 1994; Yokomori et al., 1994; Zhou et al., 1993). It does so through direct interactions
with specific transcriptional activators such as Zta (Ozer et al., 1994) , Spl , NTF-l , and
VP16 (Yokomori et al., 1994) and coactivators such as PC4 (Ge and Roeder, 1994). It
has also been proposed that TFIIA can function as an anti-repressor, through interactions
with TBP, to remove negative regulatory components present in crude TFIID
chromatographic fractions (Cortes et al., 1992; Ge and Roeder, 1994; Ma et al., 1996;
Zawel and Reinberg, 1995). Pursuant to those ideas, the [3 and 7 subunit of human TFIIA
have been found to function in antirepression, while the a subunit is required for

activation (Ma et al., 1996).

TFIIB

Transcription factor TFIIB is a single polypeptide of 35 kDa (Ha et al., 1991) that
is an essential component of the RNA polymerase II initiation complex. It enters the
preinitiation complex after TBP binding to the TATA box. TFIIB acts as a bridging
factor by stabilizing the TBP-TATA interaction on the promoter complex and recruiting
RNA polymerase II and TFIIF (Buratowski et al., 1989). Because of its bridging role,
TFIIB bears two unique structural features that reinforce its importance in preinitiation
assembly and function. TFIIB contains at its N-terminus a Zn2+ finger domain that is
dispensable for interaction with TBP, but is important for efficient recruitment of
polymerase-TFIIF and to support basal transcription initiation GBarberis et al., 1993;
Buratowski and Zhou, 1993; Hisatake et al., 1993). Mutations at the N-terminus inhibit
assembly of transcription intermediates. At the C-terrninus lies two imperfect direct

repeats similar to the core domain of cell cycle regulatory proteins (Bagby et al., 1995;

18

Gibson et al., 1994). The two cyclin-like C-terminal repeats of TFIIB (TFIIBc) are
necessary and sufficient for interaction with the TBP-TATA complex (Hisatake et al.,
1993). TFIIBc is deficient in transcription due to its inability to support efficient
recruitment Of the RNAPII/TFIIF complex (Barberis et al., 1993; Buratowski and Zhou,
1993; Ha et al., 1993; Yamashjta et al., 1993).

Based on the solved three-dimensional structure of TFIIBc (Bagby et al., 1995)
and TFIIBc-TBP-DNA ternary complex (N ikolov et al., 1995), the C-terminal region of
TFIIB binds TBP, activators (Roberts and Green, 1994) and DNA . TBP-TFIIB contacts
are mainly between the basic amino-terminal repeat of TFIIB and the acidic carboxy-
terrninal “stirrup” of TBP. Recent experiments demonstrate that TFIIB is a sequence-
specific DNA-binding protein that recognizes a novel sequence element found in certain
promoters (Lagrange et al., 1998). This interaction may stabilize the melting of the
promoter near the transcription start-site. The N-terrninus is postulated to be a scaffold
for the assembly of RNA polymerase II-TFIIF . In addition to the functional interaction
between TFIIB and TFIIF during transcription initiation, both factors cooperate with each
other to regulate dephosphorylation of the CTD of RNA polymerase II afier transcription

termination (Chambers et al., 1995).

TFIIF (RAP30/74)

By itself, RNA polymerase 11 cannot stably associate with the preinitiation
complex subassembly, and must be escorted to the promoter by TFIIF. The subunits of
TFIIF, RAP30 and RAP74, were purified from HeLa cell extract by affinity

chromatography on a column containing immobilized calf thymus RNA polymerase II as

19

a ligand (Sopta et al., 1985). Based on their affinity to bind RNA polymerase II this
group of proteins was termed RNA polymerase lI-Associated Proteins (RAPs), the
number indicating the apparent molecular weight in kDa. RAP30 and RAP74 were
shown to be required for accurate transcription initiation from several promoters, defining
the TFIIF complex as a general initiation factor that binds RNAPII (Flores et al., 1988).
Antibodies directed against RAP30 co-immunoprecipitate RAP74, indicating that RAP30
and RAP74 are normally associated in a complex (Burton et al., 1988). Both subunits Of
TFIIF are required for accurate initiation in RAP30-immunodepleted HeLa cell nuclear
extract and in vitro assay systems reconstituted with purified components (Burton et al.,
1988; Burton et al., 1986; Flores et al., 1989). TFIIF was not initially identified as one of
the four original HeLa cell chromatographic fractions (TFIIA, TFIIB, TFIID, and TFIIE)
required for accurate initiation by RNAPII (Sawadogo and Roeder, 1985). Further
purification resolved the TFIIE fraction into two factors, TFIIE and TFIIF, both of which
are essential for initiation (Flores et al., 1989). Purification of the TFIIF fraction defined
the two subunits, identical to RAP30 and RAP74 (Flores et al., 1990). In solution and in
the preinitiation complex, TFIIF is a heterotetrarner of two RAP30 and two RAP74
subunits (Conaway and Conaway, 1989; Flores et al., 1990; Robert et al., 1998). Gel
filtration studies have shown that the apparent molecular weight of the TFIIF complex is
220-280 kDa suggesting a heterotetrarneric 012132 structure (Conaway and Conaway, 1989;
Flores et al., 1990; Kitajima et al., 1990).

TFIIF homologues have been identified and purified from rat liver as initiation
factor By , from Drosophila as Factor 5, and from yeast as factor g (Conaway and

Conaway, 1989; Henry et al., 1992; Kitajima et al., 1990; Price et al., 1989). Yeast TFIIF

20

(factor g) is composed of three subunits with apparent molecular masses of 105, 54, and
30 kDa encoded by genes TF G1, TF G2, and TFG3, respectively (Henry et al., 1992).
ngl and ng2 are the functional and structural homologues of mammalian RAP74 and
RAP30 (Henry et al., 1994). ng3 represents a dispensable subunit which is less tightly
associated and slightly stimulatory for yeast TFIIF activity . The gene for TFG3 was
previously isolated as ANCl, a gene involved in cytoskeletal function (Welch et al.,
1993). ng3 has no known human counterpart but bears close sequence similarity to
human leukemia proteins ENL and AF -9 (Henry et al., 1994; Welch and Drubin, 1994).
ng3 has been identified as a TFIID subunit , yTAFu30 (Poon et al., 1995) , and is a
component Of the SWI/SNF chromatin-remodeling complex (Cairns et al., 1996).

Similar to bacterial 0 factors, TFIIF prevents spurious initiation by inhibiting and
reversing the binding of polymerase to non-promoter sites on DNA (Conaway and
Conaway, 1990; Killeen and Greenblatt, 1992). In fact both subunits exhibit limited
sequence similarity to bacterial 0' factors (Garrett et al., 1992; McCracken and
Greenblatt, 1991; Yonaha et al., 1993). It is not clear whether these structural similarities
reflect functional similarities. However, RAP30 appears to be partially analogous to
bacterial E.coli O70 . Human RAP30-RAP74 can bind E. coli RNA polymerase and can
be displaced by 0’70 (McCracken and Greenblatt, 1991). Two conserved regions have
been identified (Garrett et al., 1992; Sopta et al., 1989). Primary structure analysis of
human RAP30 revealed that the greatest sequence homology between RAP30 and E. coli
0'70 is located in the 2.1 o conserved region of 0'70 (McCracken and Greenblatt, 1991).
This region Of O70 is required for binding to E. coli core polymerase (Lesley and

Burgess, 1989). The carboxy terminus of the RAP30 has weak sequence similarity with

21

region 4 of bacterial 0' factors; in particular, it exhibits significant similarity to the
carboxyl-terminal regions 4.1 and 4.2 of SpoIIIC (Bacillus subtilis 6K) (Garrett et al.,
1992). The C-terminus of mammalian transcription factor RAP30 has been found to be a
cryptic DNA-binding domain (Tan et al., 1994). Strikingly, the DNA-binding domain of
RAP30 is homologous to 6 factor region 4, believed to direct recognition and binding Of
RNA polymerase at the -35 element of bacterial promoters (Dombroski et al., 1992). The
NMR structure of the C-terminus of human RAP30 possesses a helix-turn-helix fold,
similar to the linker histone H5 and the hepatocyte nuclear transcription factor HNF-
3/f0rk head (Grofi et al., 1998).

The human cDNAs encoding RAP30 and RAP74 (Finkelstein et al., 1992; A50 et
al., 1992) have been cloned (Aso et al., 1992; Finkelstein et al., 1992; Sopta et al., 1989).
RAP30 and RAP74 produced in E. coli can replace natural human RAP30/74 to direct
accurate transcription in vitro (Finkelstein et al., 1992). Some of the functions of TFIIF
can be carried out by RAP30 alone. The RAP30 subunit has the property of suppressing
nonspecific DNA binding of RN APII. RAP30 can deliver the polymerase to the promoter
to support transcription initiation in the absence of RAP74 (Killeen et al., 1992). In
addition, the TFIIF complex can remove RNAPII from nonspecific DNA sites, whereas
RAP30 alone can only prevent it from binding to these sites (Conaway and Conaway,
1990; Killeen et al., 1992).

In vitro evidence suggests that TFIIF binds to RNAP II primarily through RAP30.
RNAPII protects and prevents the phosphorylation of RAP30 by protein kinase A in the
polymerase core homology region to 0'70 (McCracken and Greenblatt, 1991).

Recombinant RAP30 also binds RNAPII, indicating that the RAP30 subunit directs

22

binding of TFIIF to polymerase (Killeen et al., 1992). It is also likely that a RAP30-DNA
interaction can enhance the stability of the preinitiation complex. This is consistent with
the photocrosslinking of RAP30 to sequences between the TATA box and the
transcription start site (Coulombe et al., 1994; Kim et al., 1997; Robert et al., 1996).
Although RAP30 can carry out many of the functions of TFIIF alone, the full
complement of TFIIF activities requires the presence of RAP74. RAP74 appears to play
roles in at least three stages of transcription, i.e. initiation, promoter escape, and
elongation. For initiation, RAP74 participates in TFIIF’s binding to polymerase by
stabilizing either the interaction between RAP30 and polymerase or polymerase and the
DNA (Garrett et al., 1992; Tyree et al., 1993). Indeed, RAP74 can interact directly with
RNAPII through its C-terminus, which is masked by the N-terminus and central region
(Wang and Burton, 1995). Curiously human RAP74 can enhance the binding of bacterial
RNA polymerase to a promoter and stimulate its transcription in vitro (Chibazakura et al.,
1994). RAP74 protein can be divided into three regions including an N-terminal and C-
terminal globular domain and a flexible highly charged central region (Lei et al., 1998;
Wang and Burton, 1995). Deletion mutagenesis studies have indicated that the N-
terminal regions of both RAP30 and RAP74 are responsible for their interaction and are
essential for transcriptional activity (Wang and Burton, 1995; Yonaha et al., 1993).
Genetic studies have implicated RAP74 in start site selection. A mutation in the yeast
TFIIB gene (SUA7) that causes aberrant start site selection is suppressed by secondary
mutations in the yeast RAP74 homologue (Sun and Hampsey, 1995). This thereby

restores the use of the normal start site. In support of this idea, RAP74 is able to interact

23

with TFIIB (Fang and Burton, 1996) and can be crosslinked to promoter sequences
between the TATA element and the start site (Robert et al., 1996).

RAP30 and RAP74 were initially thought to function exclusively in initiation and
in elongation, respectively, however both subunits are now known to function in both
processes (Tan et al., 1994). RAP74 has also been implicated in RNAPII promoter escape
(Chang et al., 1993). RAP74 is dispensable for initiation but is required for very early
elongation. The requirement for RAP74 in promoter escape may be due to its function in
late complex assembly. TFIIF can increase the rate of transcription elongation by
suppressing transient pausing of the polymerase (Bengal et al., 1991; Bradsher et al.,
1993; Flores etal., 1989; Izban and Luse, 1992; Price et al., 1989). The C-terrninal
domain Of RAP74 also stimulates a CTD phosphatase activity that removes phosphate
groups from the CTD of polymerase (Archambault et al., 1997; Archambault et al., 1998;
Chambers et al., 1995). This stimulation by RAP74 can be blocked by TFIIB, indicating a
dynamic interaction between TFIIF, TFIIB, and CTD phosphatase thought to be involved
in the recycling of RNAPII (Dahmus, 1996; Roeder, 1996). It has been shown that the C-
terminal domain and the central region of RAP74 are necessary for continuous RNA
initiation in an extract system, possibly by the stimulation Of CTD phosphatase activity
(Lei et al., 1998).

Photocrosslinking studies have defined the topology of a TBP-TFIIB-TFIIF-RN A
Polymerase II-TFIIE promoter complex. RAP74 and RAP30 bind promoter DNA
between the TATA box and start site, a region where TFIIE and RNAPII also crosslink
(Coulombe et al., 1994; Kim et al., 1997; Robert et al., 1996). RAP74 also binds DNA

upstream of TATA, inducing a conformational change that affects the position of

24

RNAPII relative to the DNA template and wraps the promoter DNA around polymerase
and TFIIF (Forget et al., 1997). From these studies and analysis of RAP74 deletion
mutants, a clear correlation was drawn from the activities of RAP74 fragments in
wrapping the DNA with their behavior in transcription (Lei et al., 1998; Robert et al.,
1998). Due to the wrapping of promoter DNA around polymerase in the preinitiation
complex, the DNA is first bent at the TATA box by TBP and bent again near the
transcription start site. It is postulated that the bending and wrapping cause a superhelical
distortion on the promoter that destabilizes the DNA strands near the start site. This
strand unwinding then allows the access of TFIIH helicase to separate the template
strands in the presence of ATP to form an Open complex. The DNA-wrapping model has
important implications for transcription initiation and elongation and the role of TFIIF in
converting the polymerase into an isomerized or active state.

TFIIF can undergo several covalent modifications. Both TFIIF subunits are highly
specific substrates for covalent poly ADP- ribosylation (Rawling and Alvarez-Gonzalez,
1997). The role of this modification remains unknown. RAP74 is heavily phosphorylated
in vivo, in accordance with the presence Of several potential phosphorylation sites in its
sequence (Aso et al., 1992; F inkelstein et al., 1992; Kitajima et al., 1994). RAP74 has
been shown to be phosphorylated in vitro by casein kinase 11, protein kinase A, the TFIID
subunit TAF11250 (Dikstein et al., 1996; Yonaha et al., 1997) and TFIIH (Ohkuma and
Roeder, 1994). RAP74 was recently shown to possess a serine/threonine kinase activity,
allowing an autophosphorylation of serine 385 and threonine 389. These sites are
phosphorylated by casein kinase II-like kinases and TAF11250, a component of TFIID

(Rossignol et al., 1999). The precise role of RAP74 phosphorylation has not been

25

established, although in vitro data suggest that both the initiation and elongation activities
of TFIIF are stimulated by phosphorylation (Kitajima et al., 1994). Recently it was
suggested that the CK20t and its phosphorylation of RAP74 can affect the in vivo

recruitment Of TFIIF to promoter DNA (Egyhazi et al., 1999).

TFIIE

Human TFIIE is a heterotetramer of 56 kDa (HE-0t) and 34kDa (HE-B) subunits
(Conaway et al., 1991; Inostroza et al., 1991; Ohkuma et al., 1990). However, two
dimensional crystallography of a TFIIE- RNAP II complex suggests that yeast TFIIE
may exist as an heterodimer (Leuther et al., 1996). Structurally, both subunits include

defined structural motifs. TFIIE-0t has a zinc ribbon motif, a helix-turn-helix motif, a

leucine repeat and protein kinase consensus sequences (Ohkuma et al., 1991). TFIIE-B
contains a consensus nucleotide binding site. TFIIE is essential for basal transcription. It
enters the preinitiation complex after RN AP II and TFIIF. Its incorporation during
preinitiation complex assembly is required for the recruitment of TFIIH (Buratowski et
al., 1989; Flores et al., 1992; Flores et al., 1989). TFIIE has been demonstrated to interact
physically with RNAP II, TFIIH and TFIIF (Maxon et al., 1994). TFIIE is functionally
associated with TFIIH. Other functions Of TFIIE include the stimulation of the CTD
kinase activity of TFIIH, stimulation of TFIIH dependent ATP hydrolysis (Lu et al.,
1992; Ohkuma et al., 1995; Ohkuma and Roeder, 1994) and promotion of DNA wrapping
within the preinitiation complex (Robert et al., 1998; Robert et al., 1996). Specifically,
TFIIE-cc mediates the helicase activity of TFIIH either negatively (Drapkin and Reinberg,

1994) or positively (Serizawa et al., 1994). It has been shown that TFIIE in association

26

with TFIIH and ATP hydrolysis are required for promoter clearance (Goodrich and Tjian,

1994).

TFIIH

TFIIH is a multiprotein complex consisting of nine proteins whose molecular
weights range from 34 kDa to 89 kDa (Drapkin and Reinberg, 1994; Maldonado and
Reinberg, 1995). Its yeast counterpart contains 5 subunits (Feaver et al., 1993). Recently,
a functionally active human TFIIH was reconstituted from baculovirus recombinant
polypeptides (Moreland et al., 1999). TFIIH is by far the most complex basal
transcription factor that contains at least three identifiable enzymatic activities
(Orphanides etal., 1996), including two ATP-dependent helicase (Schaeffer et al., 1993;
Serizawa et al., 1993), a DNA-dependent ATPase (Conaway and Conaway, 1989; Roy et
al., 1994) and a kinase specific for CTD phosphorylation of the largest subunit of RNAP
II (Feaver et al., 1991; Lu et al., 1992; Serizawa et al., 1992).

TFIIH plays a fundamental role in transcription because it is implicated in the
transition from transcription initiation to elongation. Part of this step is mediated by the
CTD kinase activity of TFIIH. The concept has been advanced that phosphorylation of
the CTD causes a conformational change in the preinitiation complex that facilitates
promoter escape (Goodrich and Tjian, 1994; Usheva et al., 1992). The CTD kinase
activity resides with the MOIS/Cdk7 subunit, a cyclin-dependent kinase (Feaver etal.,
1994; Roy et al., 1994). Cyclin H, a regulatory partner of Cdk7, is also present in TFIIH
(Serizawa et al., 1995). In combination with an activating protein MAT-1, the ternary

complex cdk7/Cyclin H/MAT-l forms what is called a CAK (cdk-activating kinase)

27

complex (Drapkin et al., 1996; Orphanides et al., 1996). The presence of Cak in TFIIH
suggests the possibility of a link between cell-cycle regulation and transcription that has
not clearly been established. In addition to its CTD kinase role, TFIIH also specifically
phosphorylates TBP, TFIIE-0t and TFIIF’s RAP74 subunit (Ohkuma and Roeder, 1994).

Another crucial step that affects the transition from transcription initiation to
elongation is TFIIH’s role in forming the open transcription complex (Dvir et al., 1996;
Holstege et al., 1996). TFIIH is required for transcriptional initiation from linear DNA in
vitro. The requirement for TFIIE, TFIIH, and ATP can be obviated when transcription
takes place on a DNA template that is partially unwound, such as negatively supercoiled
DNA or artificially premelted DNA (Holstege et al., 1995; Pan and Greenblatt, 1994;
Parvin and Sharp, 1993; Tyree et al., 1993). Because TFIIE contains no know enzymatic
activity, open complex formation appeared to be mediated by TFIIH DNA helicase
activity. One of the two DNA helicases in TFIIH, namely ERCC3 in human and Rad25 in
yeast, is essential for transcriptional initiation and believed to be necessary for promoter
melting to form the “open” complex (Feaver et al., 1993; Guzder et al., 1994; Guzder et
al., 1994; Park et al., 1992; van Vuuren et al., 1994). In yeast, Rad25p was also
demonstrated to be required in vivo (Moreland et al., 1999).

TFIIH’s ERCC3 subunit is identical to the XPB excision repair protein (Schaeffer
et al., 1993). XPB/ERCC3 (Excision repair cross-complement) is a helicase implicated
in the human DNA-repair disorders xeroderrna pigmentosum (XP) (Weeda et al., 1990)
and Trichothiodystrophy (TTD) (Weeda et al., 1997) . TFIIH has been proposed to play a
central role in coupling transcription and DNA repair (F riedberg, 1996; Sancar, 1996).

This role in nucleotide excision repair (N ER)/ DNA repair was established by the ability

28

of purified TFIIH to rescue the repair deficiency of mammalian and yeast TFIIH mutants

(Drapkin et al., 1994; van Vuuren et al., 1994; Wang et al., 1994).

TRANSCRIPTION BY RNA POLYMERASE II

Initiation

The general process of initiation is similar to that catalyzed by bacterial RNA
polymerases. Binding of RNA polymerase generates a closed complex, which is
converted at a later stage to an open complex in which the DNA strands have been
separated. A separate stage of the initiation reaction requires ATP hydrolysis, release of
transcription factors and polymerization of RNA. Transcriptional initiation by RNA
polymerase II proceeds through four distinct stages: PIC assembly, open complex

formation, abortive initiation, and promoter clearance (Zawel and Reinberg, 1995).

Preinitiation Complex Assembly (PIC)

In vitro experiments have established a stepwise pathway for the assembly of the
preinitiation complex (PIC) on the AdMLP promoter (Conaway and Conaway, 1990;
Flores et al., 1991; Van Dyke et al., 1988) and identified various intermediate steps that
remain stable after challenges such as nuclease-protection, gel mobility shift and template
competition assays (Buratowski et al., 1989; Van Dyke et al., 1989; Zawel and Reinberg,
1993). In addition, protein crosslinking and X-ray crystallography has established the
relative locations of DNA, RNAPII, TBP, TFIIA, TFIIB, TFIIE and TFIIF (Coulombe et

al., 1994; Forget et al., 1997; Geiger et al., 1996; Kim et al., 1993; Kim et al., 1997; Kim

29

et al., 1993; Nikolov et al., 1995; Robert et al., 1996; Tan et al., 1996; Tang et al., 1996).
The first step in the assembly of the initiation complex is binding of TFIID to the TATA
box. For basal transcription, a single TBP is sufficient for TATA element recognition and
subsequent incorporation of the other basal factors. TFIID instead of TBP is required for
initiation from TATA-less promoters possibly through DNA-TAF interactions (Martinez
et al., 1998; Weis and Reinberg, 1992). In addition, transcription regulation by upstream
regulatory factors requires the TFIID complex. Binding Of TBP to the TATA element
through minor-groove contacts forms a very stable complex and involves dramatic DNA
distortion (Burley and Roeder, 1996; Nikolov et al., 1996). The resulting bend brings
sequences upstream and downstream of the TATA closer together (Geiger et al., 1996;
Tan et al., 1996). TFIIA then joins the complex after TFIID binding, forming the “DA”
complex. While TFIIA is not essential for basal transcription with purified factors, it can
function to establish and maintain the committed complex under more physiological
conditions (Lee et al., 1992). TFIIA stabilizes the TFIID-DNA interaction through direct
contacts with both TBP and upstream DNA sequences (Geiger et al., 1996; Tan et al.,
1996) . Although the precise mechanism is unknown, TFIIA can relieve the repressive
effects of certain factors including Dr] (Inostroza et al., 1992), topoisomerase I (Ma et
al., 1996; Merino et al., 1993) and MOTl (Auble et al., 1994) . TFIIA can displace
repressors that are associated with TBP (Cortes et al., 1992; Coulombe et al., 1992; Ma et
al., 1996; Merino et al., 1993; Ranish and Hahn, 1991) and enhance TBP’s affinity for
promoter DNA (Auble et al., 1994) thereby allowing TBP to nucleate the PIC. Binding of
TFIIB, through direct interactions with TBP and with DNA sequences both upstream and

downstream of the TATA element, results in a “DAB” or “DB” complex in which the

30

direct-repeat domain of core TFIIB is oriented towards the initiation site. TFIIB serves
as a bridge between TBP and RNAPII, and this interaction is important for transcription
start site selection (Leuther et al., 1996; Li et al., 1994; Pinto et al., 1992). Similar to
TFIIA, TFIIB can stabilize artificially weakened TBP -TATA interactions (Imbalzano et
al., 1994). Binding of a pre-formed TFIIF- RNAPII complex, through direct interactions
Of TFIIB with both TFIIF and polymerase, follows the binding of TFIIB (Leuther et al.,
1996; Zawel and Reinberg, 1995) , forming a “DBPolF” or “DABPolF” complex.
Polymerase by itself is poorly recruited to the “DB” or “DAB” complex without TFIIF.
Although TFIIF plays a direct role in promoter targeting of RNA Polymerase II, it also
plays an indirect role by reducing RNAP II binding to non-specific sites in DNA (Zawel
and Reinberg, 1995). The small subunit of TFIIF, RAP30, appears sufficient for RNAP
II recruitment (Conaway and Conaway, 1993; Flores etal., 1991; Killeen and Greenblatt,
1992; Tyree et al., 1993), however genetic and biochemical studies suggest that both
RAP30 and RAP74 are necessary for stabilization of the PIC intermediate, through
TFIIB interactions (Fang and Burton, 1996; Sun and Hampsey, 1995; Tan et al., 1994).
Forming the “DABPolFE” or “DBPOIFE” complex, the next step in the pathway is the
binding of TFIIE, through its direct interactions with RN AP 11, TBP, and potentially
TFIIF (Maxon etal., 1994). TFIIE incorporation is necessary for subsequent recruitment
of TFIIH (Flores et al., 1992). Binding of TFIIH completes assembly of the
“DABPolFEH” preinitiation complex. Within the complete PIC, protein-DNA contacts
now extend to position +30 (Buratowski et al., 1989; Van Dyke et al., 1988).

Initiation can also occur in the absence of some of the general transcription

factors such as TFIIE, TFIIH and TFIIF on supercoiled templates (Parvin and Sharp,

31

1993; Tyree et al., 1993). Negative supercoiling of DNA can facilitate the unwinding of
DNA strands, and obviate the need for the TFIIH, TFIIE, and ATP hydrolysis in
promoter melting to form an open complex on linear DNA templates (Timmers, 1994).
Experiments with pre-opened heteroduplex templates indicate the dispensability of
TFIIH, TFIIE, and ATP hydrolysis (Holstege et al., 1996).

Limited studies have been performed with TATA-less (Inr) promoters and have
suggested that PIC formation may be distinct for TATA and Inr promoters (Smale, 1997).
Preinitiation complex assembly on TATA-less promoters requires specific TAFns
(Kaufrnann and Smale, 1994; Purnell et al., 1994), Inr-binding factors such as TFII-I
(Carcamo et al., 1991) and RNAPII (Roy et al., 1993). Inr-binding factors that have been
identified include YYI, TFII-I, USF and E2F. Using a particular supercoiled DNA
promoter, YYI, TFIIB and RNAPII were sufficient to direct accurate transcription
(U sheva and Shenk, 1994). In this case, YYI appears to substitute for TBP, as a factor
that binds to the core promoter through its Zinc finger and recruits the polymerase to the
initiation complex.

The PIC can also be preassembled in the form of an RNA polymerase 11
“holoenzyme” containing a subset of the GTFs, requiring only TBP, the remaining GTFs
and DNA for transcription initiation in response to activators (Koleske and Young, 1995).
This form of RNAPII holoenzyme is often referred to as the SRB/mediator-containing
holoenzyme. The SRB/mediator complex contains coactivators that physically link
activators to the basal transcription machinery. RNA polymerase II holoenzymes have
been purified from many eukaryotic organisms (Chao et al., 1996; Cho etal., 1997; Kim

et al., 1994; Koleske and Young, 1994; Maldonado et al., 1996; Ossipow et al., 1995; Pan

32

et al., 1997; Thompson et al., 1993). The subunit composition of these different
preparations differs somewhat involving the presence or absence of GTFs and
regulatory factors. Some protocols lead to the purification of RNA polymerase II
holoenzymes containing all of the GTFs (Ossipow et al., 1995; Pan et al., 1997;
Thompson et al., 1993), whereas other protocols generate holoenzymes in which TFIIF is
the only GTF that remains associated (Kim et al., 1994). Some yeast holoenzyme
preparations contain stoichiometric levels of Swi-Snf (Kim et al., 1994), whereas others
lack any detectable Swi-Snf protein (Cairns et al., 1996). Preparations of mammalian
RNAPII holoenzymes appear to contain very different components, such as
polyadenylation factors (McCracken et al., 1997), histone acetyltransferases (Cho et al.,
1998), and the breast cancer tumor suppressor BRCAl (Scully et al., 1997). The current
model to explain the presence of holoenzymes suggests that transcription activation at
many promoters involves recruitment of the transcription initiation apparatus in two steps
(Myer and Young, 1998). Recruitment of a TBP-containing complex (Lee and Young,
1998) and a holoenzyme containing the remaining GTFs would be required for
activation. Recruitment of either complex to the promoter could direct assembly of the
other. Activators are thought to target components of both complexes and/or multiple
components within a single complex. Given the large number of promoters present in
living cells and the diverse mechanisms known to regulate gene expression, transcription
initiation at some promoters could involve recruitment of components in many steps, and
initiation at other promoters could involve recruitment of the entire apparatus in a single

step.

33

Open Complex Formation

In addition to nucleotide triphosphates required for RNA polymerization,
transcription by RNAPII necessitates the use of hydrolyzable B—y bond of ATP or dATP
(Bunick et al., 1982; Jiang et al., 1993; Sawadogo and Roeder, 1984; Wang et al., 1992).
While eukaryotic RNAP I, III, and bacterial RNA polymerases do not require B—y bond
ATP hydrolysis, RNA Polymerase 11 requires it for initiation (Bunick et al., 1982; Eick
et al., 1994; Lofquist et al., 1993). RNAP 11 requires separation of the two DNA strands
prior to initiation of transcription. Specifically Opening of the DNA helix requires ATP
hydrolysis at the 0-7 phosphoanhydride bond (Jiang et al., 1993; Wang et al., 1992). This
process is known as “promoter melting” and is followed by formation of the first
phosphodiester bond of the nascent RNA. Open complex formation allows access of
nucleotide triphosphates to the template strand. TFIIH’s ATPase activity mediates the
activation of the basal initiation complex. TFIIH’s associated DNA helicase activity is
crucial for creating a single-stranded region during formation of the open complex.
TFIIH helicase activity is required for maintaining the Open region before formation of a
four-nucleotide RNA product (Holstege et al., 1997). TFIIE may also play a direct role in
promoter melting in the absence of TFIIH by stabilizing the single-stranded DNA in the
melted region (Holstege et al., 1995) by binding single-stranded DNA through its a
subunit zinc-ribbon motif (Kuldell and Buratowski, 1997). Photocrosslinking studies
indicate that TFIIE stabilizes the wrapping of promoter DNA around RNAPII and TFIIF
in the PIC (Robert etal., 1998; Robert et al., 1996). It is proposed that DNA wrapping
has a direct role for topological untwisting of the helix around the transcription start site

(Robert et al., 1998).

34

Abortive Initiation

Abortive initiation, leading to synthesis and release of oligomers up to ten
nucleotides, competes with productive initiation which leads to the formation of stable
elongating complexes. Bacterial RNA polymerases (Gralla et al., 1980; Levin et al.,
1987; Tintut et al., 1995) go through an abortive phase of transcription in which
approximately 75% of the time the short RNA is aborted and synthesis must be restarted.
Similarly, accurate initiation of productive RNA synthesis in vitro at the adenovirus
major late promoter is accompanied by abortive initiation of very short transcripts
(Bunick et al., 1982; Jacob et al., 1994; Linn and Luse, 1991; Luse and Jacob, 1987).
Abortive initiation may represent an energy barrier that RNAP II has to overcome in
order to begin productive elongation. Alternatively, abortive initiation may reflect a
conformational change that has to occur to convert the initiation complex to a productive

elongation complex (Buratowski et al., 1989).

Promoter Escape

Promoter escape is the event which results in the transition from abortive
initiation to productive elongation. This transition must break protein-protein and protein-
DNA contacts that establish the initiation complex. This transition requires a
conformational change in RNAPII (Samkurashvili and Luse, 1998) and release of some
initiation factors. RNA polymerase promoter escape in bacterial systems involves the
release Of a 0' factor and changes in polymerase conformation and template contacts
(Metzger et al., 1989). After promoter escape, TBP remains bound to the promoter,

whereas TFIIB, TFIIE, TFIIF, and TFIIH are released from the complex (Zawel and

35

Reinberg, 1995). Phosphorylation of the RNAP II CTD has long been associated with the
transition from initiation to elongation (Laybourn and Dahmus, 1990; Payne et al., 1989).
This observation has suggested a role for CTD phosphorylation in promoter clearance
(Conaway and Conaway, 1993), and the TFIIH CTD kinase activity is known to mediate
this modification (Svej strup et al., 1996). It was found in a defined transcription assay,
that TFIIE, TFIIH, and ATP hydrolysis are not required for abortive initiation, but instead
required by promoter clearance from a linear template (Goodrich and Tjian, 1994). As
described above, this requirement could be related to DNA strand separation rather than
CTD phosphorylation. The role for TFIIH in promoter clearance may explain why
helicase and CTD kinase activities are found in one complex. DNA unwinding may
stimulate promoter clearance, while CTD phosphorylation may mark polymerases that
have successfully progressed into elongation. TFIIH-mediated phosphorylation of the
CTD within the PIC has been proposed to promote conformational changes that release
RNAPII from general initiation factor contacts thought to be important for either PIC
assembly, activation or initiation steps. Interactions that are reversed by phosphorylation
include CTD-TBP and RNAPII TFIIE interactions (Flores et al., 1989; Maxon et al.,
1994; Usheva et al., 1992; Zawel and Reinberg, 1993). SRB proteins and other factors in
the RN APII holoenzyme complex that interact with the CTD might also be reversed by
CTD phosphorylation. The requirement for CTD phosphorylation by TFIIH remains
unclear. In the purified basal factor system, a role for CTD phosphorylation in promoter
clearance has not been shown for the AdML promoter (Makela et al., 1995; Serizawa et
al., 1993). However, CTD phosphorylation is required for promoter clearance at the

Adenovirus E4 (AdE4) promoter (Jiang et al., 1996). CTD kinase activity of TFIIH at a

36

post initiation phase is also required at the dihydrofolate reductase (DHFR) promoter
(Akoulitchev et al., 1995). ATP hydrolysis, therefore, may result from the TFIIH-
associated DNA helicase activities required for promoter opening. Additionally, in cell

extract systems, the RAP74 subunit of TFIIF (Chang etal., 1993) and P-TEFb (Marshall

and Price, 1995) are required for productive promoter escape.

Elongation

After polymerase has escaped from the promoter, translocation of RNAPII occurs
processively on the DNA as it enters the elongation phase during which the transcription
complex synthesizes nascent RNA. The synthesis reaction is powered by free energy
liberated by nucleotide polymerization. The elongation stage of RNAPII is a complex
process that involves a number of regulatory factors and is increasingly being recognized
as a major regulatory process of gene expression (Bentley, 1995; Greenblatt et al., 1993;
Spencer and Groudine, 1990; Uptain et al., 1997; von Hippel, 1998; Wright, 1993). Well
documented examples of transcription regulated at the elongation level include
oncogenes c-myc, c-fos, and c-myb (Kerppola and Kane, 1991), HIV (Greenblatt et al.,
1993) and Drosophila hsp70 (Lis and Wu, 1993).

Messenger RNA synthesis in eukaryotic cells can take place at rates of 1200-
2000 nucleotides per minute (Tennyson et al., 1995; Thummel et al., 1990; Ucker and
Yamamoto, 1984), while elongation by purified mammalian RNAPII in vitro proceeds at
rates of only 100-300 nucleotides per minute (Izban and Luse, 1992). Detailed
mechanistic studies have shown that purified RNAPII is highly susceptible to sustained

pausing and arrest during elongation on naked DNA and chromatin templates (Aso et al.,

37

1995; Izban and Luse, 1992; Uptain et al., 1997). Promoter-proximal pausing is released
by DNA- or RNA-binding activators that recruit or stimulate positive-acting transcription
elongation factors. Release Of RNAP II from stalled complexes is a rate-limiting step in
transcription Of some inducible eukaryotic genes (Izban and Luse, 1992; Kerppola and
Kane, 1991; Tennyson et al., 1995). Taken together, these findings suggest the existence
of a class of factors that stimulate the overall rate of elongation of eukaryotic mRNAs by

suppressing pausing and arrest by RNA polymerase 11 (Bentley, 1995).

Pausing, Arrest, and Termination

Throughout the elongation phase, RNA polymerase can encounter blocks that
result in transcriptional pauses, transcriptional arrest, or transcript termination (Landick,
1997). Although multiple classes Of pause, termination, and arrest signals are identified in
bacterial systems, the signals in eukaryotes are poorly understood (Landick, 1997; von
Hippel, 1998). Transcriptional pause and arrest signals can be intrinsic, a result of the
RNA polymerase’s interaction with sequences in the nascent transcript and the DNA
template (Reines et al., 1987). During a transcriptional pause, RNA polymerase
temporarily stops RNA synthesis for a finite period of time before spontaneously
resuming transcript elongation. The molecular basis for arrest is not well understood, but
a connection has been suggested between arrest and a transient failure of the polymerase
to translocate along the template. An arrested elongation complex is unable to efficiently
resume transcript synthesis without the aid of accessory factors. These factors can cause
RNA polymerase to backtrack its catalytic center to an internal phosphodiester in the

nascent RNA chain. Throughout both pauses and arrests, RNA polymerase remains

38

catalytically active, stably bound to the DNA template, and retains the nascent transcript.
Paused complexes and arrested complexes have distinct conformations (Izban and Luse,
1993). Pausing appears to be a universal prerequisite for termination (Bentley, 1995).
This effect is true for p-dependent termination in bacteria; however, not all pauses result
in termination. Whether pausing is a necessary prerequisite for all termination events is
unknown. Terminator signals cause the polymerase to irreversibly release RNA and DNA
from the ternary complex. RNA polymerases can also undergo blocks to elongation in
response to DNA binding proteins that physically hinder the progression Of ternary
complexes or under artificial conditions in which one or more of the four nucleotide

substrates is not available.

Elongation Factors

Arrested elongation complexes have not terminated, but they can only resrune
chain elongation very slowly. Protein factors that influence transcript elongation have
been isolated from human, yeast, rat, and Drosophila and have been separated into two
classes (Reines et al., 1996). The first, TFIIS (SII/RAP38) helps RNAPII to read-through
the template when it becomes arrested in ternary complexes (RNA-DNA-Protein)
because of transcriptional blocks (Reinberg and Roeder, 1987). TFIIS has been shown to
interact both genetically and biochemically with RNAPII (Agarwal et al., 1991;
Archambault et al., 1992; Sopta et al., 1985).The RNAPII subunit, pr9, appears to
specifically interact with 811 (Awrey et al., 1997). The interaction of TFIIS with the
arrested RN APII stimulates the cleavage of nascent RNA from the 3’ end, causing the

polymerase to move backwards (Izban and Luse, 1992; Izban and Luse, 1993; Johnson

39

and Charnberlin, 1994; Powell et al., 1996). This RNA cleavage reaction is stimulated by
GreA or GreB in bacteria and by 811 in eukaryotes (Borukhov et al., 1992; Borukhov et
al., 1993) . The cleavage reaction is carried out by the RNAPII itself, releasing a short
RNA fragment of 7-17 nucleotides long (Rudd et al., 1994). Because of this endonuclease
activity of RNAPII, SII can increase the fidelity of transcription (Jeon and Agarwal,
1996; Yoon et al., 1998). Transcription restarts from the newly exposed 3’ end and
RNAPII moves through the block.

A second class of structurally unrelated elongation factors include TFIIF, Elongin
(S111), ELL& ELL2 (Shilatifard et al., 1997; Shilatifard et al., 1996), DSIF (Selby and
Sancar, 1997) and C88 (Selby and Sancar, 1997).These factors increase the overall
elongation rate of RNAP by suppressing its the transient pausing (Bengal et al., 1991;
Bradsher et al., 1993; Bradsher et al., 1993; Izban and Luse, 1992; Price et al., 1989;
Shilatifard et al., 1997; Shilatifard et al., 1997 ; Shilatifard et al., 1996). As with $11,
these proteins can bind to RNAPII but function in a distinct manner because they cannot
release polymerase from arrest or promote polymerase backtracking.

TFIIF is unique among the basal transcription factors in that it has roles in both
initiation and elongation (Bengal et al., 1991; Izban and Luse, 1992; Kephart et al., 1994;
Lei et al., 1998; Tan et al., 1994). TFIIF can decrease the duration of pausing by RNAPII
and protects the elongation complex from arrest (Gu and Reines, 1995). It has been
proposed that stimulation of elongation is potentiated through the phosphorylation of the
RAP74 subunit (Kitajima et al., 1994). The kinase responsible for RAP74
phosphorylation in vivo has not been identified. A number of protein kinases are capable

of phosphorylating RAP74 in vitro. They include casein kinase 11, protein kinase C & A,

40

(Kitajima et al., 1994), TAF11250 (Dikstein et al., 1996) and RAP74 , which can weakly
autophosphorylate itself (Rossignol et al., 1999). Analysis with RAP74 mutants have
suggested that only the N-terminal of the protein is required for elongation (Kephart et
al., 1994) whereas a RAP30 central region is required for elongation (Tan et al., 1995).

The Elongin complex (SIII) functions through a direct interaction with elongating
polymerase and suppresses pausing by increasing the time polymerase spends in an active
conformation (Bradsher et al., 1993; Moreland etal., 1998). Elongin is a heterotrimer
composed of a transcriptionally active A subunit of 110 kDa and two smaller positive
regulatory B and C subunits of 18 and 15 kDa respectively (Aso et al., 1996; Takagi et
al., 1996). A role for the elongin BC complex in oncogenesis was brought to light by the
discovery that elongin B and C are components Of a multiprotein complex containing the
product of the von Hippel-Lindau (VHL) tumor suppressor gene (Duan et al., 1995; Kibel
et al., 1995). VHL disease is an autosomal dominant familial cancer syndrome that
predisposes affected individuals to a variety of tumors. Like elongin A, the VHL protein
contains a consensus elongin BC box (Kibel et al., 1995). Binding Of elongin A and VHL
to the elongin BC complex is mutually exclusive and VHL is capable of inhibiting the
activity of the elongin complex in vitro by binding tightly to the elongin BC complex and
preventing it from interacting with elongin A (Duan et al., 1995).

P-TEFb is a unique elongation factor because it promotes elongation by RNAPII
possibly through phosphorylation of the CTD (Marshall et al., 1996). P-TEFb is a
heterodimer composed of 43 and 120 kDa subunits. It was originally identified from
Drosophila as a factor important for the production of DRB-sensitive transcripts in vitro

(Marshall and Price, 1995). DRB is a nucleotide analog which has been shown to be a

41

potent inhibitor of eukaryotic mRN A synthesis in vivo and in crude transcription systems
in vitro (Chodosh etal., 1989; Marshall and Price, 1992; Zandomeni etal., 1983).
Drosophila and human P-TEFb are capable of phosphorylating the CTD of RNA
polymerase 11 (Marshall et al., 1996; Peng et al., 1998; Peng et al., 1998). The CTDs of
actively elongating polymerases are highly phosphorylated (Bartholomew et al., 1986;
Cadena and Dahmus, 1987; O. Brien et al., 1994; Payne et al., 1989). Polymerases
containing hypophosphorylated CTDs preferentially enter the preinitiation complex
(Chesnut et al., 1992; Laybourn and Dahmus, 1990; Lu et al., 1991; Serizawa et al.,
1993), where they are subsequently phosphorylated during or shortly after initiation
(Laybourn and Dahmus, 1990; O. Brien et al., 1994). DRB inhibits phosphorylation of
the CTD by P-TEFb which correlates with DRB-induced inhibition of elongation by
RNA polymerase II. P-TEFb has also been implicated in the HIV-1 Tat-mediated

transcriptional activation (Fujinaga et al., 1998; Zhu et al., 1997).

Transcriptional Termination And Recycling

The mechanism controlling RNAPII termination is poorly understood.
Termination results when RNAPII stops its processive elongation and releases the
nascent RNA (Kerppola and Kane, 1991). E. coli RNA polymerase recognizes specific
sites in the DNA template which serve as termination signals. This recognition for the
most part occurs independently of accessory factors and can occur intrinsically by
prokaryotic RNA polymerases. However, protein factors, such as bacterial rho protein,
are also required at some sites in prokaryotes. RNAPII requires release factors

downstream Of the polyadenylation addition signal. The most notable of eukaryotic

42

release/termination factors is the Drosophila N-TEF protein. The Drosophila N-TEF
protein and its human counterpart function in an ATP-dependent manner to induce
release of transcripts synthesized by RNA polymerase 11 (Liu et al., 1998; Xie and Price,
1997; Xie and Price, 1996). One of the major eukaryotic termination factors is the 3’
processing machinery for RNA polymerase II transcripts (Proudfoot, 1989). Termination
Of the nascent transcripts is coupled to processing in ways that are not understood.
Accurate and efficient termination of transcription requires the sequences and machinery
required for proper 3’end formation (Pandey et al., 1994; Proudfoot, 1989).

After polymerase has terminated and been released, it is free to reassemble the
preinitiation complex at the same or a distinct TFIID bound promoter. The same
polymerase molecule is re-delivered to a promoter and initiates another round of
transcription, a process called recycling. The polymerase must be made competent to
efficiently re-enter into the PIC. RNAPII binds to the preinitiation complex preferentially
with its CTD in the underphosphorylated RN APIIa form (Lu et al., 1991). Transition
from a preinitiation complex to an elongation complex is accompanied by
phosphorylation of the CTD, yielding to the RNAPIIO form. To recycle the polymerase,
there must be a conversion from 110 to Ila, facilitated by a phosphatase. A CTD
phosphatase that catalyzes the dephosphorylation of RNA Polymerase II has been
isolated (Chambers and Dahmus, 1994; Chambers and Kane, 1996). Polymerase
recycling may be regulated by the coordinated activities of at least TFIIF, TFIIB, and the
CTD phosphatase in a multimeric complex. The C-terrninal domain Of the RAP74
subunit Of TFIIF has been shown to strongly stimulate the CTD phosphatase (Chambers

et al., 1995). RAP74 mutants containing only the N-terminal domain do not stimulate the

43

CTD dephosphorylation and are incapable of transcriptional reinitiation (Lei et al., 1998).
However, TFIIB binding to RAP74 blocks the RAP74-dependent stimulation of the CTD
phosphatase activity (Chambers et al., 1995). The inhibitory effect of TFIIB on CTD
phosphatase activity may serve to prevent premature dephosphorylation during

productive elongation, which could cause premature termination.

TRANSCRIPTIONAL REGULATION

Transcriptional Activation

Under normal conditions, the minimal protein apparatus required for accurate
transcription initiation consists of GTFs and RNAPII. This transcription is not dependent
on the presence of regulatory factors and is known as “basal” transcription.
Transcriptional activation of eukaryotic genes during development or in response to
extracellular signals involves the regulated assembly of multiprotein complexes on
enhancers and promoters. DNA packaged into chromatin results in general repression of
gene transcription (Hanna Rose and Hansen, 1996). Activated transcription is a process
by which an activator protein induces an increase in RNAPII transcription of a certain
gene. Activators are specific regulatory proteins that bind DNA sequences located
upstream of the core promoter to ultimately increase the rate of transcription. Activators
can stimulate various steps along the pathway for mRN A synthesis and may serve to
counteract repression and increase transcription. These can include : 1) removal of
repressor molecules from promoters by recruiting, for example, chromatin remodeling

enzymes (Kingston et al., 1996), 2) recruitment of GTFs and RNAPII to the promoter to

44

facilitate PIC assembly (Paranjape et al., 1994; Ptashne and Gann, 1997), 3) stimulation
of promoter clearance and elongation (Bentley, 1995; Triezenberg, 1995), 4) induction of

covalent modifications such as phosphorylation of proteins in the PIC.

Activators

Protein fusion experiments have defined two modular functional regions of
cellular activators (Brent and Ptashne, 1985; Ptashne, 1988). One “promoter-targeting”
region or DNA-binding domain directs the protein specifically to the DNA, enabling the
other “activating domain” to stimulate transcription (Mitchell and Tjian, 1989).
Activators have loosely been classified according to the most prevalent amino acid of
their activation domains. Activators have been termed acidic (VP16, GAL4, GCN4),
glutamine-rich (Spl , Oct-l, Oct-2, Jun), proline-rich (CTF /NF1), or serine/threonine-rich
(Triezenberg, 1995). However, a pattern of bulky hydrophobic amino acid residues in
these activation domains may be more important than the prevalence of characteristic
amino acids (Cress and Triezenberg, 1991; Gill et al., 1994; Triezenberg, 1995). It is
likely that interaction and cohesion of activation domains and their targets is driven by
hydrophobic forces. Structural information of activation domains has been difficult to
obtain because these domains largely appear to be unstructured and have no secondary
structure (Shen et al., 1996). However, activation domain have been known to undergo an
induced fit, assuming more constrained structures in the presence of certain GTFs (Shen
et al., 1996; Uesugi et al., 1997).

The DNA-binding domains of activators reveal canonical protein motifs for

recognizing specific DNA sequences, including the helix-tum-helix motif, Zn-fingers,

45

and the leucine-zipper motif (Harrison, 1991). The overall potency of a transcriptional
activator depends on a number of factors: the affinity for its site on the DNA and the
subunit interactions necessary to assemble a functional activator, as well as the strength

Of the interaction between an activation domain and its target.

Transcriptional Coactivators

The ability to reconstitute activator dependent transcription in vitro with
biochemically defined components has enabled a search for partners that interact with
specific activation domains. Transcriptional coactivators, also known as mediators or
adapters are required for transcriptional activation to transduce the signal to the basal
machinery by bridging the interaction between gene-specific activator proteins and GTFs
(Berger et al., 1990; Kelleher et al., 1990; Pugh and Tjian, 1990). These factors are
distinct from activators in that most do not directly bind DNA and none appears to bind
DNA in a sequence-specific manner (Hampsey, 1998). Coactivators can also facilitate
chromatin remodeling (Kaiser and Meisteremst, 1996). Several functionally distinct
classes of coactivators have been described. These include the TAP components of TFIID
(Goodrich and Tjian, 1994), the SRB/mediator complex that associates with RNAPII’s
CTD (Hengartner et al., 1995), TFIIA, SAGA and related complexes that catalyze
nucleosomal histone acetylation (Grant et al., 1998), and the SWI/SNF and related

chromatin-remodeling complexes (Chiba et al., 1997).

46

Template Activation

Chromatin presents a sizable barrier to the process of RNA transcription,
inhibiting both the accessibility of the general transcription machinery to promoter
sequences and the binding of upstream regulatory proteins. In vitro experiments have
established that RNAPII and the GTFs cannot efficiently bind promoters packaged into
nucleosomes (Paranjape et al., 1994). Promoter binding would require the assistance of
factors that alter or remodel nucleosomes to allow access to the promoter. This
derepression would cause the chromatin structure to be decondensed and unfolded, or
modify the nucleosome components so as to affect their availability or structure. One
candidate for this activity is the multiprotein SWI/SNF complex, which has been
implicated in chromatin remodeling by genetic and biochemical studies (Peterson, 1996).
SWI/SNF can facilitate TBP binding to promoter DNA and activator binding to upstream
regulatory sequences (Cote et al., 1994; Imbalzano et al., 1994; Peterson and Tamkun,
1995). Various subunits Of SWI/SNF have been associated with both yeast and human
RNAP holoenzymes (Liao et al., 1995; Neish et al., 1998; Wilson et al., 1996).Yeast
RNAPII holoenzyme containing SWI/SNF has been shown to remodel nucleosomes
(Gaudreau et al., 1997). The SWI/SNF complex requires ATP hydrolysis for its role in
transcription stimulation and possesses a DNA-dependent ATPase activity. Interestingly,
the SWI/SNF complex possesses a weak sequence specific DNA-binding activity, that
may suggest a role for targeting RNAPII holoenzyme to specific promoters (Quinn et al.,
1996). It is unlikely that SWI/SNF is the universal nucleosome remodeler, in light of
other identified remodeling activity, such as NURF and RSC (Cairns et al., 1996;

Tsukiyama et al., 1995; Tsukiyama and Wu, 1995).

47

Chromatin mediated repression of transcription can be alleviated by the addition
of transcriptional activators such as GAL4-VP16, Spl and PHO4 (Croston et al., 1991;
Karnakaka et al., 1993; Lorch et al., 1992; Svaren et al., 1994; Workman et al., 1991).
Transcriptional activators are directly involved in the modification Of chromatin to
derepress its effect on gene transcription. Activators may recruit chromatin-remodeling
complexes to specific promoters to alter the local nucleosomal structure and allow
assembly of the PIC.

Histone acetylation is also involved in nucleosomal disruption (Struhl, 1998;
Vettese-Dadey et al., 1996). Acetylation of histone makes chromatin less condensed and,
therefore, more accessible to the transcription machinery (Hebbes et al., 1988). Two
yeast transcription coactivators, ADA and SAGA complexes, contain the specific histone
acetyl transferase GCN5 (Grant et al., 1997; Horiuchi et al., 1995). TAFu250 is a subunit
of the basal transcription factor TFIID that possesses histone acetyltransferase activity.
TAF11250 can interact with both activators and basal factors (Imhof et al., 1997; Ruppert
and Tjian, 1995; Wang et al., 1997). Transcriptional activators appear to work
synergistically with chromatin remodeling complexes such as SWI/SNF and HAT

complexes to facilitate derepression.

Recruitment of GTFs

Activator proteins work to enhance the rate at which GTFs assemble to form the
preinitiation complex and/or induce its isomerization to an activated form. Direct
interactions between activators and several components of the basal transcription

machinery (TFIIA, TBP, IIB, IIE, IIF, and IIH) have been detected (Hori and Carey,

48

1994; Zawel and Reinberg, 1995). In this model, activators bind to upstream DNA
sequences and assist the assembly of the PIC through interactions with GTFs and by
looping the DNA to permit the interaction (Kingston and Green, 1994). Alternatively,
recruitment may function to retain some GTFs at the promoter after initiation (Zawel et
al., 1995). By interacting directly with promoter bound GTFs, activators may prevent
their dissociation to facilitate reinitiation.

The association of TBP with the promoter has been characterized as being slow in
vitro (Schmidt et al., 1989). In vivo studies with the yeast acidic activator GCN4 have
suggested that TBP-TATA binding is rate limiting and activation domains can increase
the recruitment of TBP to promoters (Klein and Struhl, 1994). Similar, experiments using
GAL4-TBP fusion protein show that TBP recruited by the GAL4 DNA-binding domain
to promoters bearing a GAL4-binding site can interact with the TATA element and direct
high levels of transcription in vivo (Majello etal., 1998; Xiao etal., 1995). TBP has been
shown to bind in vitro to many activators including VP16, Spl, Oct-1, Oct-2, Gal4, C-
Jun, c-Myc, E2F1, Zta, Tat, and p53 (Chang et al., 1995; Emili et al., 1994; Franklin et
al., 1995; Ingles et al., 1991; Liu et al., 1993; McEwan et al., 1996; Melcher and
Johnston, 1995; Pearson and Greenblatt, 1997; Truant et al., 1993; Zwilling et al., 1994).
Activators may also enhance the stability of the TBP-TATA binary complex (Chen et al.,
1993). Activators can also recruit TFIID by interacting with TAF subunits (Carrozza and
DeLuca, 1996; Chen et al., 1994; Defossez et al., 1997; Gill et al., 1994; Goodrich et al.,
1993; Jacq et al., 1994; Klemm et al., 1995; Mazzarelli et al., 1997; Sauer et al., 1995;

Schwerk et al., 1995).

49

The association of the TBP to DNA promoters is a rate-limiting step in gene
expression. It was recently suggested that slow promoter binding might be related to
TBP's ability to occlude its DNA binding domain through TBP-TBP dimerization
(Coleman and Pugh, 1997). In fact, TBP and TFIID can exist as dimers in solution
(Coleman and Pugh, 1997; Jackson-Fisher et al., 1999; Taggart and Pugh, 1996). TBP
dimerization may be a mechanism to prevent unregulated gene expression and its own
degradation (J ackson-Fisher et al., 1999). This suggests that the purpose of certain
activators may be to disrupt TBP dimerization in order to enhance the rate of TBP-DNA
binding. Pursuant to this idea, VP16 has been found to induce a conformational change
in TBP that affects TBP binding to DNA (Liljelund et al., 1993). Recently, TFIIA has
also been shown to regulate TBP/TFIID monomerization and accelerate the kinetics of
DNA binding by TBP (Coleman et al., 1999).

TFIIA is dispensable for basal transcription but is required for activator mediated
transcription in vitro and in vivo (Ma et al., 1993; Ozer et al., 1998; Ozer et al., 1994).
Recruitment of TFIIA by certain activators can promote DA complex formation and help
overcome the slow step Of TBP binding in PIC formation (Chi and Carey, 1996; Chi and
Carey, 1993). Activators such as VP16, Zta, and T-antigen bind directly to TFIIA, and
the binding correlates with their ability to enhance TFIIA-TFIID-TATA complex
formation (Damania et al., 1998; Kobayashi et al., 1995; Kobayashi et al., 1998; Ozer et
aL,1994)

Transcription activation is thought to entail conformational changes in the
preinitiation complex prior to initiation of RNA synthesis. The presence of an activator

can dramatically alter the Dnase I footprint Of TFIID on a promoter (Burley and Roeder,

50

1996). It has been suggested that VP16 may induce a conformational change in TFIIB
that facilitates the binding of TFIIB to the TBP-DNA complex by disrupting
intramolecular interactions within TFIIB and stimulates RNAPII/TFIIF recruitment
(Hayashi et al., 1998; Roberts and Green, 1994). TFIIB mutants defective in activated,
but not basal transcription have been identified (Roberts et al., 1993). Indeed, TFIIB can
interact with several activators such as VP16, Gal4-AH, CTFl, Gal4, RXRB, CREB,
HIV-1 Vpr, and Vitamin D receptor (Agostini et al., 1996; Blanco et al., 1995; Choy and
Green, 1993; Kim and Roeder, 1994; Leong et al., 1998; Lin et al., 1991; Roberts et al.,
1993; Wu etal., 1996; Xing etal., 1995).

The roles of TFIIF and TFIIE in transcriptional activation are not clear. TFIIE
interacts with the Epstein-Barr virus activator EBNA 2 through a coactivator p100 (Tong
et al., 1995). The RAP74 subunit of TFIIF has been known to interact with
transactivation proteins serum response factor (SRF), androgen receptor, and VP16
(McEwan and Gustafsson, 1997; Zhu et al., 1994). A correlation of SRF-RAP74 binding
and transcriptional activation suggests that RAP74 is a critical target for SRF-activated
transcription (Joliot et al., 1995). Such a correlation has not been defined for androgen

receptor and VP16.

Stimulation of Promoter Escape and Elongation

Initiation is not the only stage of RNAPII transcription that is stimulated by
activators. As described above, post initiation steps such as promoter clearance and
elongation are also subject to regulation. Stimulation of promoter clearance not only

enhances the rate of RNA synthesis but also facilitates the entry of a second polymerase.

51

The formation of an open complex requires the ATP-dependent DNA helicase activity of
TFIIH. Binding of activators to TFIIH or TFIIE may stimulate the helicase activity and
therefore facilitate formation of the open complex.

The complete preinitiation complex contains at least two CTD specific protein
kinases, including TFIIH and the mediator/SRB complex of the holoenzyme (Liao et al.,
1995). Phosphorylation of the CTD is required for the polymerase to enter an elongation
mode. It is conceivable that activators could increase the rate of initiation by promoting
CTD phosphorylation. For example, upon the activity Of a heat shock activator HSF near
the promoter of Drosophila hsp70, enhanced phosphorylation of the CTD can induce
promoter proximal escape Of the polymerase (Lis and Wu, 1993; O. Brien et al., 1994; O.
Brien and Lis, 1991; Rasmussen and Lis, 1993). TFIIH binds activator proteins VP16,
p53, RAROL, and Tat (Garcia-Martinez et al., 1997; Rochette-Egly et al., 1997; Xiao et
al., 1994). Mutations in the VP16 activation domain have been shown to affect formation
of the open complex (J iang et al., 1994). RAROt and p53 activation domains can be
phosphorylated by TFIIH and the phosphorylation correlates with their activities in
transcription (Lu etal., 1997; Rochette-Egly et al., 1997). The Tat protein is a
transcriptional activator that is required for efficient human immunodeficiency virus 1
(HIV-1) gene expression . The interaction of Tat with TFIIH stimulates CTD
phosphorylation and activation of HIV-1 transcription (Garcia-Martinez et al., 1997;
Parada and Roeder, 1996; White etal., 1992). Interestingly, a coactivator responsible for
Tat transactivation, Tat-SF, binds and co-immunoprecipitates with TFIIF’s small subunit,
suggesting a role for RAP30 as a target in controlling elongation by Tat (Kim et al.,

1999).

52

Activators may also target known elongation factors such as P-TEF b, TFIIF, 811,
$111, and ELL. These factors act by stimulating the catalytic rate Of RNA synthesis
(TFIIF, S111, and ELL) or by facilitating the passage of RNAPII through various arrest
sites (811). Activators could stimulate elongation by recruiting these factors to the
polymerase and affecting the processivity of the elongation complex. The recent
implication that TFIIF serves to form the Open complex by inducing promoter DNA to
wrap around the PIC presents another potential target for activators (Coulombe and

Burton, 1999; Robert et al., 1998).

53

CHAPTER 2

INTERACTION BETWEEN TRANSCRIPTION FACTORS
TFIIF AND TFIIB

54

INTRODUCTION

The general transcription factor TFIIB plays an essential role in RNAPII
transcription, and together with RN APII and TBP, defines the minimal set of factors
necessary for in vitro promoter-dependent transcription Of a supercoiled DNA template
(Orphanides et al., 1996). TFIIB is involved in the selection Of transcription start sites, as
mutations in the Saccharomyces cerevisiae TFIIB homologue SUA 7 gene (Pinto et al.,
1992) alters transcription start site selection in vivo and in vitro (Bangur et al., 1997;
Pinto et al., 1994). TFIIB acts as a bridge between promoter-bound TFIID/TBP and
RNAPII/TFIIF. In both ordered-assembly and holoenzyme-recruitment models of
preinitiation complex formation, TFIIB recognizes promoter-bound TFIID and facilitates
the assembly of the remaining GTFs and RNAPII. Consistent with this role, TFIIB
interacts with DNA adjacent to the TATA box (Lagrange et al., 1998) and binds to TBP
(Buratowski et al., 1989; Ha et al., 1993; Maldonado et al., 1990), the TBP-associated
factor TAFu40 (Goodrich et al., 1993), RNAPII (Bangur et al., 1997; Bushnell et al.,
1996; Ha et al., 1993), and both subunits of TFIIF (Fang and Burton, 1996; Ha et al.,
1993). TFIIB may also play a role in the regulation of transcription by gene-specific
regulatory proteins, as many of these regulatory factors bind TFIIB directly (Chiang et
al., 1996; Colgan et al., 1995; Franklin et al., 1995; Hadzic etal., 1995; Lin et al., 1991;
MacDonald et al., 1995; Sauer et al., 1995; Xing et al., 1995).

TFIIB is comprised of a highly conserved N-terminal region and a C-terrninal
core domain. It is a 33 kDa polypeptide that contains two imperfect repeats encompassing
the C-terminal two-thirds of the molecule and an N-terminal zinc binding motif. The

C-terminal domain forms a protease resistant core (cIIB) (Barberis et al., 1993; Malik et

55

al., 1993) that interacts with the N-terminal domain (nIIB) (Roberts and Green, 1994). In
addition to the zinc binding domain, nIIB includes a conserved sequence that links the
zinc binding domain to cIIB (Na and Hampsey, 1993). Intact TFIIB has evaded structural
analysis, however crystallographic structural data have been obtained for the C-terminal
domain of human TFIIB (N ikolov et al., 1995). NMR structures have been obtained for
the core domain of human TFIIB and for a portion of the N-terminal region of the TFIIB
from archaebacteria Pyrococcusfixriosis (Bagby et al., 1995; Zhu et al., 1996).

The solution structure for human cIIB (Bagby etal., 1995), and a co-crystal
structure for a TATA-TBP-cIIB ternary complex (N ikolov et al., 1995), revealed that the
core C-terminal region is mainly or-helical, arranged in pseudo-dyad symmetry. The end
of the first direct repeat forms a basic amphipathic a helix while the second direct repeat
contains a surface cluster of hydrophobic amino acids and a helix-tum-helix motif that
interacts with specific DNA sequences located upstream of the TATA box (Lagrange et
al., 1998; Qureshi and Jackson, 1998). The free structure of cIIB is similar to its
TATA-TBP-cIIB co-crystal counterpart, except that free cIIB is more compact and the
relative orientation of the two repeats is different, suggesting a TFIIB conformational
change upon the assembly of the ternary complex (Hayashi etal., 1998).

NMR structural data have been obtained for the N-terminus of Pyrococcusfirriosus
TFIIB, revealing a zinc ribbon B-sheet similar to that present in the elongation factor
TFIIS (Zhu et al., 1996). The crystal structure of the TFIIB C-terminal domain in a
complex with TBP bound to DNA (N ikolov et al., 1995) has long suggested that the N
terminus of TFIIB would project downstream toward the region Of transcriptional

initiation and may interact with TFIIF to recruit RNAPII. The high degree of amino acid

56

conservation Of the N-terminal region Of TFIIB suggests that important functions reside
in this portion of the protein.

Biochemical and structural evidence have suggested that the N- and C-terminal
domains of native TFIIB are engaged in an intramolecular interaction (Bangur et al.,
1997; Hayashi et al., 1998; Roberts and Green, 1994). Presumably in order to form a
productive PIC, TFIIB must undergo a conformational change that would disturb the
intramolecular interaction, thereby exposing domains that are required for further
assembly of the PIC. A conformational change in TFIIB may facilitate the interaction
between TFIIB and RNAPII/TFIIF.

Interestingly TFIIB has an intimate relationship with TFIIF as part of a CTD
dephosphorylation complex. As mentioned in the previous chapter, phosphorylation and
dephosphorylation Of the CTD by the regulated activities of CTD kinases (F eaver et al.,
1991; Lu et al., 1992; Marshall et al., 1996; Serizawa et al., 1992; Serizawa et al., 1993)
and phosphatases (Chambers and Dahmus, 1994)) appear to control progression through
the transcription cycle. A CTD phosphatase that catalyzes the dephosphorylation of
RNAPII has been shown to interact (Archambault et al., 1997; Archambault et al., 1998)
and is stimulated by the C-terminal domain of the RAP74 subunit of TFIIF (Chambers et
al., 1995). RAP74-dependent stimulation of CTD phosphatase activity is blocked by
addition of TFIIB. The C-terrninal domain of RAP74 binds to TFIIB (Fang and Burton,
1996) and RNAPII (Wang and Burton, 1995); therefore, TFIIF, TFIIB, and the CTD
phosphatase may be components of a multiprotein complex that binds RN APII and
regulates the polymerase recycling mechanism, affecting the pool of competent

polymerases available for transcription of genes.

57

From amino acid sequence (Aso et al., 1992; F inkelstein et al., 1992), RAP74 is
proposed to have highly basic N- and C-terrninal domains separated by a highly charged,
overall acidic, and flexible central region that is rich in charged amino acids, E, D, K, and
R, and also S, T, P, and G (Figure 1). The N-terminal domain is sufficient to support
preinitiation complex assembly, single-round initiation, and elongation by RNAP II. The
central region and C-terminal domain of RAP74 have a small stimulatory effect on
initiation but their most important function is in multiple-round transcription in vitro.
Deletion Of the central region or just the C-terminal domain of RAP74 creates a protein
with partial function in multiple-round transcription (Lei et al., 1998). Multiple-round
transcription is likely to represent a recycling mechanism for RNAPII or an essential
polymerase transcription factor. In an effort to map the domain of RAP74 responsible for
multiple-round transcription and to elucidate the role of the TFIIB-TFIIF interaction in
transcription, we have generated and characterized a collection of RAP74 mutants in in
vitro transcription, gel mobility shift and protein-protein interaction assays. Using surface
plasmon resonance, we have mapped the domain of RAP74 responsible for interaction
with TFIIB. In marked contrast to previously published results (Barberis et al., 1993;
Buratowski and Zhou, 1993), we also show that the N-terminus Of TFIIB is not required

for the recruitment of RNAPII and TFIIF in vitro.

58

Figure 1. N- and C-terminal domains Of RAP74, which were originally proposed from
sequence analysis, correspond to distinct functional domains. The N-terminal domain
has most of the required functions for single-round initiation and elongation. The central
region and C-terrninal domain of RAP74 function in multiple-round transcription.
Regions shaded dark are very important for activity. Key: IIB) TFIIB; CTDP) CTD

phosphatase; PIC) preinitiation complex.

59

 

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60

MATERIALS AND METHODS

Transcription Factors and Extracts.

Recombinant human RAP30, RAP74, and RAP74 mutants were prepared and
quantitated as described (Fang and Burton, 1996; Wang and Burton, 1995; Wang et al.,
1993; Wang et al., 1994). Calf thymus RNAPII used in electrophoretic mobility shift
experiments was prepared by the method of Hodo and Blatti (Hodo and Blatti, 1977) and
was primarily in the IIb form, lacking the carboxy terminal domain (CTD). Recombinant
yeast TBP and human recombinant TFIIB were the kind gifts of Steven Triezenberg and
Fan Shen. The clones for production Of wt TFIIB and TFIIB truncation mutants were
the kind gifts of Danny Reinberg, James Geiger, and Masarni Horikoshi. The clones for
production of human TFIIEG. and TFIIEB were kindly provided by Dr. Danny Reinberg.
Recombinant TFIIE subunits were produced in E. coli, purified, and TFIIE complexes
were reconstituted as described (Maldonado et al., 1996). TFIIB mutants were purified
as described and stored in 1M guanidine HCl (Hisatake et al., 1993).

Human HeLa cells were purchased from the National Cell Culture Center
(Minneapolis, MN). Extracts of HeLa cell nuclei were prepared as described (Shapiro et
al., 1988). A TFIIF-depleted extract was prepared by immunoprecipitation Of TFIIF with
anti-RAP30 and anti-RAP74 antibodies (Burton et al., 1988; F inkelstein et al., 1992).
The TFIIF-depleted extract was completely dependent on the re-addition of RAP30 for

activity and was strongly stimulated by addition Of RAP74.

61

RAP74 Mutagenesis and Recmitution of Recombinflt TFIIF

RAP74 mutants, l-492,1-472, and 1-450, were constructed with appropriate
primers by polymerase chain reaction amplification of a plasmid clone encoding RAP74
and subcloning between the NdeI and XhoI sites of pET21a (N ovagen). Site-directed
mutants of human RAP74 were constructed using the Quick Change Site-Directed
Mutagenesis Kit (Stratagene) and appropriate primers. Double and triple alanine and
charge reversal mutagenesis was done in the 470 to 517 region of RAP74 to identify
regions of the C-terminal domain that are important for transcriptional function. The
following are the RAP74 point mutation mutants that were constructed : LL473AA,
KK475EE, KK480EE, EQT487AAA, VL492AA, IL496AA, KR498EE, LNP500AAA,
RK504EE, M1506AA, MHF511AAA. Mutated RAP74 genes were confirmed by DNA
sequencing. RAP74 mutants and the RAP30 constructs have a C-terminal six histidine
tag. Recombinant proteins were overexpressed in E. coli BL21(DE3) and purified to near
homogeneity from inclusion bodies by affinity chromatography on Qiagen Ni-NTA resin
in buffer containing 4M urea. (Wang et al., 1993; Wang et al., 1994). The TFIIF
complex was formed by adding equimolar amounts of RAP30 to each RAP74 mutant
protein. Urea was removed by dialysis into high salt buffer lacking urea to renature and
reconstitute the complex. Protein concentration was determined by absorbance at 280 nm

using calculated extinction coefficients.

Multiple-round and Single-round Transcription Assays

Transcription was initiated from an adenovirus major late promoter template

digested with SmaI to produce a +217 base runoff transcript. A 20 ul reaction mixture

62

consisted Of 0.8 pg adenovirus major late promoter DNA and TFIIF-depleted
transcription extract (72 ug total protein) supplemented with recombinant RAP30 and
RAP74 or a RAP74 mutant (10 pmol each), in transcription buffer (12 mM HEPES, pH
7.4, 12% glycerol, 0.12 mM EDTA, 0.12 mM EGTA, 1.2 mM DTT) containing 60 mM
KCl and 12 mM MgC12. For all reactions, preinitiation complexes were formed for 60
min at 30 °C. For multiple round transcription, 600 uM ATP, CTP, GTP and 25 uM
0132P -UTP (10 uCi per reaction) were added and transcription continued for 60-90
minutes. 0.25 % sarkosyl was added afterwards to block new initiation, and transcription
was continued for an additional 30 min to complete all previously initiated chains
(Hawley and Roeder, 1985). For the single-round sarkosyl block assay (Hawley and
Roeder, 1985), 600 pM ATP, CTP, and 25 uM [01-32P]UTP (10 uCi per reaction) were
added and incubated for 1 min. 600 uM GTP and 0.25 % w/v sarkosyl were added and
transcription continued for 59 min. 0.05 % sarkosyl was previously shown to be
sufficient to block new initiation by RNAPII (Hawley and Roeder, 1985), so sarkosyl
was added in five-fold excess over the amount necessary to constrain transcription to a
single round. Reactions were stopped by addition of 200 III 0.1 M sodium acetate pH 5.4,
0.5 % sodium dodecyl sulfate, 2 mM EDTA, and 100 ug/ml tRNA, followed by phenol-
chloroform extraction and ethanol precipitation. Samples were electrophoresed in a 6%
polyacrylamide gel containing 50 % urea (w/v). Dried gels were analyzed by
autoradiography and the yield of the accurately initiated transcript was quantitated using

a Molecular Dynamics phosphorimager.

63

Transcription with Purified Components

A transcription system consisting of purified calf thymus RN APII, recombinant
human TBPc, TFIIA, TFIIB, TFIIE, TFIIF, and a negatively supercoiled DNA template
was modified from (Malik etal., 1998; Parvin and Sharp, 1993). The template was the
plasmid pML(C2AT)A71, a gift of Michele Sawadogo, containing the adenovirus major
late promoter fused to a G-less cassette at position +11 (Sawadogo and Roeder, 1985;
Sawadogo and Roeder, 1985). A 20 ul reaction mix, typically composed of 0.3 pmol of
calf thymus RNAPII, 0.8 pmol TBPc, 1 pmol TFIIB, 1 pmol TFIIE, 1 pmol TFIIF, 1
pmol TFIIA, 200 ng of supercoiled pML(C2AT)A71, 20 pg BSA, and 2 U RNase
inhibitor (Promega) was assembled at room temperature. The buffer contains 60 mM
KCl and 6 mM MgC12. 600 uM ATP, CTP, and 25 uM [or-32P]UTP (5 uCi per reaction)
were added to each reaction and reactions were stopped after 1 hr incubation at 30 0C.
Transcripts were isolated and analyzed on a 6% polyacrylamide gel containing 50% urea.

Dried gels were analyzed by autoradiography.

Electrophoretic Mobility Shift Assay

The DNA probe for the gel shift assay was the adenovirus major late promoter
from position -53 to +14 relative to the transcriptional start site. The probe was
synthesized by the polymerase chain reaction using the primers: 5'-32P-
CAGGTGTTCCTGAAGG-3' and 5'-ATGCGGAAGAGAGTGA-3'. The upstream
primer was radiolabeled using y-32P -ATP and T4 polynucleotide kinase. Afier
amplification, the DNA probe was gel-purified using the Qiaex kit (Qiagen). Mobility

shifts were performed according to Wang and Burton (Wang and Burton, 1995) with

64

some modifications. The reaction mixtures (15 ul) contained 20 mM HEPES pH 7.9, 20

mM Tris pH 7.9, 50 mM KCl, 2 mM DTT, 0.5 mg/ml BSA (bovine serum albumin), 10%

v/v glycerol, radiolabeled DNA probe, and proteins, incubated at 30 0C. Saccharomyces
cerevisiae TBPc (0.6 pmol) was combined with the DNA template (approximately 40
fmol) for 15 min. Recombinant human wild type or mutant TFIIB (0.6 pmol) was then
added and incubated for 15 min. Calf thymus RNAPII (0.3 pmol) was incubated with
human recombinant wt or mutant TFIIF (1 pmol) for at least 5 min prior to addition to the
DB complex. RNAPII was incubated with TFIIF and TFIIE (where indicated) for 5 min
before addition to DB and further incubation for 30 min. Reaction mixtures were loaded
onto a 4% polyacrylamide gel containing 0.09 % bisacrylarnide, 2.5 % glycerol and 0.5X

TBE (tris-borate-EDTA). Dried gels were analyzed by autoradiography.

Biosensor Kinetic Measurements.

The binding affinities of the TFIIF mutants with TFIIB were determined using a
BIACORE 2000 real time kinetic interaction analysis system (Biacore, Inc., Piscataway,
NJ) to measure association 0(a) and dissociation (k4) rates (Schuck, 1997).
Carboxymethylated Dextran biosensor chips (CM5) were activated with EDC [N-ethyl-
N’-(3-dimethylaminopropyl) carbodiimide hydrochloride] and NHS (N -
hydroxysuccinimide) according to supplier instructions. For immobilization, recombinant
TFIIB or TFIIF mutants in 10 mM sodium acetate, pH 4.5 were injected onto the
biosensor chip at a concentration Of 20 ug/ml to yield approximately 400-700 RU’s
(resonance response units) of covalently coupled protein. Unreacted groups were blocked

with an injection of 1M ethanolarnine. Kinetic measurements were carried out in binding

65

buffer (10 mM HEPES, pH 7.9, 150 mM NaCl, 0.005 % Surfactant P20) by injecting
serial dilutions of recombinant proteins at 25°C using a flow rate of 15 III/min.
Association (k) and dissociation (k4) rates were calculated using the 1:1 Langmuir
association kinetic model in the BIACORE evaluation software. The equilibrium

dissociation constant (K0) was calculated as ka/kd.

66

RESULTS

Multiple- And Single- Round Transcription With RAP 74 Truncation Mutants Reveal The
Importance Of The RAP 74 C-Terminus.

The RAP74 N-terminal domain extending from a.a. 1 to 217 supports most
RAP74 firnctions for preinitiation complex assembly, accurate initiation, and elongation
stimulation. RAP74(1-217) had a specific defect in multiple- but not single-round
transcription and this defect was not attributable to degradation or inactivation of the
mutant protein. Deletion of just the C-terrninal and/or central regions of RAP74 creates a
protein with partial function in multiple-round transcription. The region of RAP74
between a.a. 409-517 has been found to be important for multiple-round transcription
(Lei et al., 1998). The central region between a.a. 217-356 was weakly able to stimulate
multiple-round transcription in the absence of the C-terminal domain.

An important question that remains to be answered is what C-terrninal sequence
between a.a. 409-517 contributes to multiple round transcription. Implicit with this
question is whether the effect on multiple round transcription is due to a defect in
initiation, elongation, promoter escape, recycling of polymerase through decreased CTD
phosphatase stimulation, TFIIB binding, or polymerase binding. To determine what
region in the C-terrninal domain is important for multiple round transcription, RAP74(1-
492), RAP74(1-472), RAP74(1-450) truncation mutants were constructed, reconstituted
with RAP30 to form TFIIF (Figure 2), and tested for the ability to support single- and
multiple- round transcription in vitro.

RAP74 C-terminal mutants 1-492, 1-472, and 1-450 all showed significant

defects in multiple round transcription (Figure 3B). RAP74 (1-492) mutant, which

67

Figure 2. SDS-PAGE of TFIIF C-terminal truncation protein complexes stained with
Coomassie blue : lane 1, 2 rig/band protein marker standard; lane 2, wild type TFIIF
[30/74(l-517)]; lane 3, TFIIF (1-492); lane 4, TFIIF(1-472); lane 5, TFIIF(1-450).
Histidine-tagged recombinant RAP30 and RAP74 were overexpressed in E. coli
BL21(DE3) and purified to near homogeneity from inclusion bodies by affinity
chromatography on Ni2+-NTA resin in buffer containing 4M urea. The TFIIF complex
was formed by adding equimolar amounts Of RAP30 to each RAP74 mutant protein. Urea

was removed by dialysis into high salt buffer lacking urea to renature and reconstitute the

complex.

68

 

 

TFIIF C-terminal Truncation Mutants

a“ 10‘ a?“
S St» S
V
M $0 4 0 §

 

69

represented a truncation of 25 amino acids, was significantly reduced for multiple round
transcription. This truncation reduced the total transcription signal tO 38 % of wt TFIIF
activity. Further truncation to 1-472 and 1-450 (data not shown) did not significantly
aggravate the defect, indicating the importance Of amino acids 492-517. Multiple round
transcription for RAP74 C-terminal mutants 1-492, 1-472, and 1-450 did not show
significantly higher activity than the 1-217 mutant. As previously characterized, TFIIF
RAP74(1-217) mutant was shown to be defective for multiple round transcription but
shows near maximal activity for single round transcription compared to wt RAP74
(Figure 3A). This mutant serves as a control for comparing multiple and single round
defects that may be present in RAP74 mutants. RAP74 C-terminal mutant 1-492 did not
show a significant defect in single round transcription as its activity approximated both
wt RAP74 and RAP74(1-217) levels (Figure 3A). The activity of 1-472 and 1-450 (data
not shown) in multiple round transcription was below 50% of wt TFIIF activity. These
results corroborated previous indications that the C-terminal domain of RAP74 stimulates
multiple round transcription (Lei et al., 1998) and further define sequence within the

domain that is important for this activity.

Biosensor Measurements Of T FIIF, RAP30, And RAP 74 Binding T0 T FIIB.

RAP74, TFIIB, and the CTD phosphatase represent essential players in
polymerase recycling. Polymerase enters the preinitiation complex with its CTD in the
Na state. Progression through elongation is stimulated by CTD phosphorylation by CTD
kinases. In order for the polymerase to re-enter the transcription cycle after termination,

the CTD must become dephosphorylated. It has been demonstrated that the C-terminal

70

Figure 3. Single and Multiple Round Transcription with TFIIF C-terminal truncations 1-
217, 1-492, and 1- 472. In vitro transcription was done with a TFIIF depleted Hela
nuclear extract (DE). Transcription was initiated from the adenovirus major late promoter
digested with restriction endonuclease Smal tO produce a +217 base runoff transcript. 5
pmol of TFIIF complex was used for all reactions. Preinitiation complexes were allowed
to form for 60 min at 30 °C. Panel A) for the single-round sarkosyl block assay, ATP,
CTP, and radiolabeled UTP were added and incubated for 1 min. GTP and 0.25 %
sarkosyl were added and transcription continued for 60 min. Reinitiation was blocked by
sarkosyl. Panel B) for the multiple-round assay, ATP, CTP, and radiolabeled UTP were
added. ATP, CTP, GTP and radiolabeled UTP were added and transcription continued for
60 minutes. 0.25 % sarkosyl was added afterwards to block new initiation, and
transcription was continued for an additional 30 min to complete all previously initiated
chains. Samples were electrophoresed in a 6% polyacrylamide gel containing 50 % urea

(w/v). The accurately initiated transcript was quantitated using a phosphorimager.

 

 

DE,DNA,30
74 or mutant St
, ACU’ 59 min Op
Single Round ,7 l I // I

1 \
G/sarkosyl

 

 

60 min ACIGU‘ 60 min 30 min 31°F
Multiple Round I/I ' // K // :
sarkosyl

Single Round Assay
120%
110% « 100%

100% . A

90%
80%
70%
60%
50%
40%
30%
20%
10%

0%

 

% Transcription

 

 

 

thFllF 1-217 1-492 1-472

Multiple Round Assay

 

100%. .1°°% B

% Transcription
8.
a~

30% ~
20% <
10% 1
0% -

  

 

 

 

wt'l'Fll= 1-217 1-492 1-472
TFIIF Complex

Figure 3

72

domain Of RAP74 is able to bind TFIIB and polymerase and stimulate the CTD
phosphatase. TFIIB antagonizes stimulation of the phosphatase by RAP74. Evidence
seems to suggest that TFIIB and TFIIF cooperate tO modulate the interconversion of the
two forms Of polymerase.

Wild-type and mutant TFIIF proteins were assayed for stable association with
TFIIB, because changes in how these two proteins interact could affect polymerase
recycling and provide clues to explain defects in multiple round transcription (Figure
38). Binding kinetics was determined using a BIACORE biosensor system, a machine
designed for real-time interactive biomolecular interaction analysis (Schuck, 1997). The
detection principle relies on an Optical phenomenon that detects changes in the refractive
index of the solution close to the surface of the sensor. The on and off rates of the
interaction are measured and a binding constant is calculated. Concentration-dependent
binding studies were conducted tO allow calculation of kinetic parameters. Non-linear
analysis of the association and dissociation parts Of the sensorgrams showed good curve
fitting to the 1:1 (A + B c: AB) association model. The A + B <:> AB association model
was used tO determine the dissociation binding constant for TFIIF, RAP74, and RAP30
binding to TFIIB. Both TFIIF subunits are capable of binding TFIIB individually. RAP30
binding to TFIIB yielded a KB of 1.49 x 10'7 M (Figure 4 A) while RAP74 binding to
TFIIB had a dissociation constant of KD = 4.22 x 10’7 M (Figure 4 B). The TFIIFzTFIIB
binding constant had a KB of 1.43 x 10'8 M (Figure 5). Both TFIIF subunits have
dissociation constants within the same magnitude, however the TFIIF complex binds
TFIIB with a 3.4 to 10 fold higher affinity. TFIIB-TFIIF contact is largely maintained

through RAP74. It is possible that a conformational change occurs in RAP74

73

Figure 4. Binding kinetics analysis of TFIIF subunits RAP30 and RAP74 binding
immobilized TFIIB. The physical interaction Of both RAP30 and RAP74 with TFIIB was
measured by surface plasmon resonance using the BIACORE biosensor. The test ligand
was immobilized on a CM5 sensor chip by amine coupling. Concentration-dependent
binding studies were conducted to allow calculation Of kinetic parameters. Non-linear
analysis Of the association and dissociation parts Of the sensorgrams showed good curve
fitting to the 1:1 (A + B <:> AB) association model. The A + B <:> AB association model
was used to determine the dissociation binding constant (KD) for RAP30 and RAP74 with
TFIIB as indicated on the figures. Interaction of TFIIF with Bovine Serum Albumin
protein was used as control to subtract non-specfic interactions that may occur between

TFIIF and the carboxymethyl matrix on the chip.

74

 

Response

' sud
. ,, r\-"“‘
,A‘N‘ I

 

 

RAP30 Binding TFIIB

A

KD =1.49 x 10-7 M

.'\a
\ "
v '(--‘. , , .
M ' --.. ‘V"‘-"
l ., .. - . .I- 1‘

_... w
' ...W‘W , "drama“.

AW ' . W: '5 \ "\"li‘V' ~w’u
" - ' av“ ,_ ,

m-v‘fiK‘ 4r Rt“ Yaw 1, “NM-L“
I E WM

\ “of aw-N‘H‘V”
~. I .

 

.
T““"..\~‘—\‘ — qvfl~

H‘" “V. 4"“ ' u“ WM
‘7 Z’MQJI-Y‘N‘Y.‘ {M ‘
i . 4 .AM- ,v'n...‘ fi-M‘ M“~"¥W,i.v-9«

84*
e
o
6,
8
2‘;

Tim.

»-~500rIMRAP3O ~400nMRAi=30 —-—~300nMRAP30 ~———200nMRAP30

 

 

RAP74 Binding TFIIB

 

135 i B
1
115 - '4
if T
K = 4 22 x 10'7 M
95 ‘i ..r" ' ' U
”1' r ’ D
i ’9'. I‘
I" 1...;- .
, .30 '
' ...! . ,
- 1,... .. .
75 '0 /’ J/f’ \ .
-M .
f, ' , u
l , r . \ \
J I, ’\ \‘_ ~
, I ...-v , ‘ a -\
55 43 l ,’ I’M-fl .1 \_ \ A.
1 .«' “'4‘. \ \ ‘
_. ‘- ~,/“ 1 \ w“
A \
l I /. . \\ “. ‘. ‘Os
, I, , ‘- ..\ -._ m . ...~.
l I’ / I'- ' - ' \‘_\ \‘—.‘. | TT". «... ‘ i
35-“ : a: I t/ 5“. l T‘- '-‘ ‘.’T-—-. hit 'T"{u ‘T
. .‘ . ... I T'-‘--.‘ ..~‘
l; .7] V\‘\“‘~" . -. -'-~\—,, "K.“ -‘~‘~“'-“’—.'~./T
{,1 - A‘. M“~-}.“.~ “1, ...
’- ‘ N v , V ‘ fi".4'.le’
.‘r ""“~ :
.5. 11* M ~~-w....i......,--
A *— jl 2
'5 iTT'I'i i I 1': 7 T‘I I'I i'ifir'irrrr'r'i'r'i‘r I'i i'i'?’r'ifr*1*1*r*r'rrr T'i'r'r T'i‘TTTfiT'iTT'I i I I

 

-1(X)-&J-60-40-20 00 30 50 70 90110130150170190210230250270290310330350370390

2001M RAP74 118

TI".

- 175nM RAP74 118 _, --. 1501M RAP74 llB 125nM RAP74 118 -----~ 1m RAP74 "8

Figure 4

75

410 430
a

Figure 5. Binding kinetics analysis Of wild type TFIIF binding immobilized TFIIB

protein. Determination of kinetic parameters was identical to Figure 4.

76

 

“Eu: 2Com ll “Eu—... Zcov

“Eu—H 2Com , ...—....t. Scam

05:.

omv omv can own can 80 EN ovm o 5

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(I’DS/i ul‘rba I . A J ..\.'n 5.! a
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47.2.{4'} r 31.3%..
.5 3 .
(v

21: x 22 ”ex

 

. 11.....(rlr T4... . n .
(f. .. S T. /\/1 3p (5:? .4. z . \ /r;.\1r.;... «ltir/rl . I ‘3

if. i 4 (17¢..(>))\.L at)...’
W.)Ju‘. ) \‘
. . . ...: >1: ... 1 a 1..
_ .51 J... 11.34.......s,f.sa.....§)u..?.,. (48L. ....

...
. . u \(b( .9 \eu-q \
t e 1 A, s. 107...; r r .
’ . ) ... e Ks... « ...) .ffk c ......J .............:).i\.i.. 1.. c . \t

. . .b IPA. I
.\o. iii—k ‘11-?!)‘115!’.\(.\\Ih1..r~ (C‘. I K \u‘.

r

a .K 5») led {.(oif?2.\5$.. ., . , , ., . .. . .
_ ’9‘ <S ‘1)?3‘f)‘. j > .( . , . . t
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may wanna ...—Eh

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Figure 5

77

Figure 6. Structural and functional domains Of TFIIB and SDS-PAGE Of TFIIB
truncation mutants stained with Coomassie blue. Two TFIIB deletion mutants, TFIIB-N
(1-113) and TFIIB-C (114-316), were used to determine which domain interacts with
TFIIF. TFIIB has two domains : a 192 amino acid C-terminal region containing two
imperfect direct repeats, and a 124 amino acid cysteine rich N-terminal region which
contains a zinc—ribbon domain. The end of the first direct repeat forms a basic
amphipathic OI helix. The C-terminal direct repeats are important for TBP-DNA
interaction and basal transcription. By contrast, the N-terminus is dispensable for
formation of the DB complex but is required for basal transcription initiation. SDS-
PAGE of TFIIB truncation mutants: Lane 1) 2 ug/band protein marker standard. Lane 2)

wild type TFIIB. Lane 3) TFIIB- N. Lane 4) TFIIB-C.

78

 

Human TFIIB

‘___, A Basal Transcription

 

D-B-DNA Complex Forrpati_<)#n___>

1 15 37 124 200 218 294 31s
++++
1 15 37 113
114124 200 218 294 316

++++

TFIIB-N
TFIIB-C

Q
E
'—

   

Figure 6

79

as a result of its association with RAP30 that allows it to bind TFIIB with much higher
affinity, as is seen with the intact TFIIF complex. However, this scenario does not

preclude RAP30’s participation in this interaction.

T FIIF binds the N-terminal domain of TFIIB (1-113)

In addition, the dissociation constant for TFIIF binding to the N- and C- terminus
of TFIIB was determined using TFIIB truncation mutants. Two TFIIB deletion mutants,
TFIIB-N (1-113) and TFIIB-C (114-316), were used to determine which domain of
TFIIB interacts with TFIIF (Figure 6). These mutants segregate the protein into two
domains containing the Zinc finger and the direct repeat sequence responsible for
interaction with the TBP-TATA complex. TFIIB bears two unique structural features that
reinforce its importance in preinitiation assembly and fimction. TFIIB contains at its N-
terrninus a Zn2+ finger domain that is dispensable for interaction with TBP, but is
important for efficient recruitment of polymerase-TFIIF and to support basal transcription
initiation. The N-terminal domain Of TFIIB binds the RAP30 subunit of TFIIF and RNA
polymerase 11. At the C-terminus lies two imperfect direct repeats that are necessary and
sufficient for interaction with the TBP-TATA complex. BIACORE analysis revealed that
TFIIF interaction with TFIIB takes place at the N-terminus of TFIIB (Figure 7A). The
TFIIF:TFIIB-N binding indicates slightly higher affinity than TFIIF :TFIIB with a KD of
1.25 x 10‘8 M. The C-terminal repeats of TFIIB do not appear to interact with TFIIF
based on the flat sensorgrarn from the interaction between TFIIF:TFIIB-C (Figure 7B).
The structure of the TATA -TBP-TFIIBc complex strongly indicated that the interaction

between TFIIB and TFIIF might be through the N-terminal region of TFIIB (Nikolov et

80

al., 1995). The TFIIF:TFIIB-N BIACORE analysis corroborates the inference previously
made based on the three dimensional structure of TFIIB-C. A summary of the

dissociation binding constants (K9) is presented in the table below :

 

 

 

 

 

 

Binding Interaction KD -
TFIIF : TFIIB 1.43 x 10" M
TFIIF : TFIIB-N 1.25 x 10‘8 M
TFIIF ; TFIIB-C NO binding
RAP30 : TFIIB 1.49 x 10'7 M
RAP74 : TFIIB 4.22 x 10'7 M
Table 1

RAP 74 Region 492-517 Is Required For TFIIB Interaction With TFIIF.

In the absence of the RAP30 subunit, RAP74 C-terminal deletion mutants 1-492,
1-472, and 1-450 were all able to tightly bind TFIIB, although the response was slightly
lower than full length RAP74(1-517) (Figure 8A). TFIIF complexes with wild type
RAP30 and RAP74 mutants were also tested for TFIIB binding. When RAP74 mutants 1-
492, 1-472, and 1-450 are complexed with RAP30, none , however, were able to bind
TFIIB, as indicated by the flat sensorgram responses (Figure 8B). Truncation of the first
C-terminal 25 amino acids significantly reduced or eliminated TFIIF binding to TFIIB.
These results seem to indicate that, at least in vitro, TFIIB and TFIIF contact is mediated
by the C-terrninal region of the RAP74 subunit . The region between RAP74 a.a. 492 and
517 is required for this interaction. Even though RAP30 is able to bind TFIIB, RAP30
does not seem to contribute to TFIIB binding when it is associated with RAP74 as

measured with BIACORE.

81

Figure 7. Kinetic analysis Of TFIIF binding immobilized TFIIB-N (1-113) and TFIIB-C
(114-316). Panel A) TFIIF interaction with TFIIB takes place at the N-terminus of TFIIB
Panel B) The C-terminal repeats Of TFIIB do not appear to interact with TFIIF, based on
the flat sensorgram from the interaction between TFIIF:TFIIB-C . The structure Of the
TATA -TBP-TFIIBc complex strongly indicated that the interaction between TFIIB and
TFIIF might be through the N-terminal region of TFIIB. The TFIIF:TFIIB-N BIACORE
analysis corroborates the inference previously made based on the three dimensional

structure of TFIIBc.

82

 

3o_.
E 25 —-

 

TFIIF Binding TFIIB-N

..~ s MW“... ,
Am" W" ‘ “""V‘wwfi. - i
u! T'Mh‘ :;
\ A...“ 5.
r' _ '3
l “,
' e

 

#

 

 

  

i rfrfii i‘ii“f'+'rri'iriri'i'T'i
160 200 220 240 260 290 320 340 360 390 400 420 440 400 490 500
Time s
——IomMTFnF —80nMTFilF —60nMTFilF ------40rMTFilF —20nMTFIIF
RU TFIIF Binding TFIIB-C
207

Response

 

 

 

 

 

10 aniF

100 nM IIF ~--- 60 nM llF — 40 nM IIF 20 nM llF

Figure 7

83

Figure 8. Qualitative BIACORE analysis of RAP74 and TFIIF truncation mutants
complexes binding to immobilized TFIIB. Panel A) In the absence of the RAP30
subunit, RAP74 C-terminal deletion mutants 1-492, 1-472, and 1-450 were able to bind
TFIIB with the same apparent affinity as wild type RAP74. Panel B) When complexed
with RAP30, the RAP74 region between a.a. 492 and 517 is required for TFIIF-TFIIB
interaction, as indicated by the flat sensogram responses. TFIIF mutants (1-492, 1-472,
1-450) were unable to bind TFIIB. These results seem to indicate that TFIIF and TFIIB

contact is largely mediated through the RAP74 C-terminus.

84

 

RAP74 Binding TFIIB .. TFIIF Binding TFIIB

 

 

 

 

 

 

 

 

RAP74(1-492) Binding TFIIB TFIIF(1-492) Binding TFIIB
:2 -. 1
no. / m 3:
‘21 l .. 1
at T ‘ .. I- I ~-- _ W “—
RAP74(1-472) Binding TFIIB TFIIF(l-472) Binding TFIIB
3103 mi

 

 

RAP74(1-450) Binding TFIIB TFIIF(l-450) Binding TFIIB

It

 

 

 

 

’50 - 200 1
:00 < I"
,z.eemmr— “W I
150 . '_,- -" ' '
//’ 100 4
it!) I 1
\‘\~.
'\
l l l ..
«H l
o < ”4.x.” 0 4 ~ w
40 V T 40 7 v T
-& an O Q :0 In I” no 80 210 are on so 70 too no .0 .0 220 260
The The

Figure 8

85

RAP 74 C-terminal Mutagenesis.

There is a clear indication that RAP74 sequence from 493 to 517 is important for
multiple round transcription. Truncation of 25 amino acids at the C-terminal end reduced
the total transcription level by more than 50 % (Figure 3B) and compromised TFIIF’s
ability to bind TFIIB. Detailed mutagenesis within this region was undertaken to reveal
critical residues that are important for transcriptional function and TFIIB interaction.
Alignment of the C-terminus of human RAP74 with homologues from Drosophila and
yeast indicates that the region is very highly conserved among all three species (Figure
9). PHD secondary structure prediction analysis (Rost et al., 1994) indicates that the
region encompassing a.a. 470 to 517 Of human RAP74 may be comprised of two or-
helices that are likely to be preserved in RAP74 homologues. Hydrophobic cluster
analysis (HCA) is a method that relies upon a two-dimensional representation of protein
sequences for comparison and analysis of distantly related proteins when no three-
dimensional data is available (Gaboriaud et al., 1987). Hydrophobic cluster analysis
(HCA) was used to indicate amino acid clusters of likely importance for C-terminus
function Of RAP74. Double, and triple amino acid substitutions are indicated beneath the
sequence. Triple alanine and charge reversal mutagenesis in the 475 to 517 region was
used as a screen to identify regions of importance in the C-terminal domain. A
combination of alanine substitutions and charge-reversal mutations was constructed
based on the notion that these mutations would cause changes in activity without
inducing long-range changes in protein conformation. These mutants were subsequently

assayed in transcription assays and monitored for TFIIB interaction.

86

Figure 9. Alignment of human, Drosophila, and S. cerevisiae of the C-tenninal region of
RAP74. Shaded residues indicate conserved amino acids. PHD secondary structure
prediction analysis indicates that the region encompassing a.a. 470 to 517 of human
RAP74 may be comprised of two OL-helices that are likely to be preserved in RAP74
homologues. A combination of alanine substitutions and charge-reversal mutations in the
475 to 517 region was used to identify regions Of importance in the C-terminal domain.
The substitutions are indicated beneath the sequence and are summarized in the bottom

table.

87

 

 

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mm mm mm mm
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88

TE 118- TE 11F interaction Is Not Important for Multiple-Round Transcription.

The objective for doing the site-directed mutagenesis was to find specific RAP74
mutants that would be severely compromised for initiation or would at least mimic the
multiple round transcription defect seen with the RAP74 C-terminal deletion mutants.
This would establish a relationship or correlation between TFIIF mutants compromised
for TFIIB interaction and a specific defect in multiple round transcription (Figure 3B),
thereby assigning a functional assay for TFIIF C-terminal mutants. RAP74 C-terminal
deletion mutant (1-492) supports a single round Of transcription approximately z 60 % of
native TFIIF (Figure 3A). All Of the C-terminal point mutants were able to support a
single round Of transcription within a range of 60-80 % of native TFIIF, roughly similar
to 1-492 (Figure 10 A). We had hoped that detailed mutagenesis within RAP74 sequence
493 to 517 (important for TFIIF:TFIIB interaction) would reveal a few critical residues
that are important for supporting multiple round transcription. Surprisingly, most of the
point mutants affect multiple round transcription to varying degrees (Figure 10 A). Many
of the mutants were moderately defective in both single and multiple round transcription,
however the pattern was not clear which made the interpretation of these results
complicated. None Of the defects in multiple round transcription seen with these mutants
was as drastic as transcription levels reported previously (less than 50%) for the C-
terrninal deletion mutants (1-493 and 1-472). Two mutants, KK480EE and EQT487AAA,
supported maximal or near maximal levels of transcription compared to native TFIIF
(Figure 10 A).

Based on the premise that defects in multiple round transcription may be due to

compromised TFIIBzTFIIF binding, the assay reveals that TFIIBzTFIIF interaction is not

89

Figure 10. Single and Multiple Round Transcription with TFIIF C-terrninal point

mutants. In vitro transcription was done with a TFIIF depleted Hela nuclear extract (DE).

Transcription was initiated from the adenovirus major late promoter digested with
restriction endonuclease Smal to produce a +217 base runoff transcript. 5 pmol of TFIIF
complex was used for all reactions. Preinitiation complexes were allowed to form for 60
min at 30 C’C. Panel A) For the single-round sarkosyl block assay, ATP, CTP, and
radiolabeled UTP were added and incubated for 1 min. GTP and 0.25 % sarkosyl were
added and transcription continued for 60 min. Reinitiation was blocked by sarkosyl.
Panel B) For the multiple-round assay, ATP, CTP, and radiolabeled UTP were added.
ATP, CTP, GTP and radiolabeled UTP were added and transcription continued for 60
minutes. 0.25 % sarkosyl was added afterwards to block new initiation, and transcription
was continued for an additional 30 min to complete all previously initiated chains. F517
and F217 represent wild type TFIIF and TFIIF(1-217), respectively. Samples were
electrophoresed in a 6% polyacrylamide gel containing 50 % urea (w/v). The accurately

initiated transcript was quantitated using a phosphorimager.

90

 

Multiple Round //

DE,DNA,30

 

74 or mutant
ACU‘ 59 ' Stop
Single Round //_l_l§___//m_m__
G/sarkosyl
60/min ACIGU‘ 60 min 30 min Step
- // 'fi/r—l
sarkosyl
Single Round Assay

110%

100% -
90% - ,
80% -

 

 

100%

  

 
  

 

 

 

 

g 70%« 7 57% 69% 53%
§ 60% - I ' ’
g 50% ~ '
F 40% ~ .
3‘ 30% - ~-
20% - ‘
10% — .
0% - '
’\ ’\ <9 <9 to <9
,4 ,4 game gee ée‘gqx’gev’ge bgovtbgegovfif
\>’ 43‘ 43‘ 00‘ 4" 5' § 9* \t g»
Q/ ‘1‘
Miltiple Round Assay
110%
100% 93% 101%

% Transcription

 

 

 

Figure 10

91

important for multiple round transcription. Most likely, the defects in multiple round
transcription that we see involve more than simple disruption of TFIIBzTFIIF interaction.
The C-terminus of RAP74 is also involved in RNA polymerase II binding (Wang and
Burton, 1995) and CTD phosphatase stimulation (Chambers et al., 1995). Compromised
TFIIF: CTD phosphatase interaction could affect the pool Of competent RNAPII
available for re-initiation (recycling) from a promoter. Changes in the affinity of TFIIF

for RNA polymerase 11 could affect both initiation and elongation.

Reconstituted Transcription Assay With Purified Components And RAP74 C-terminal
and Point Mutants.

The initiation transcription assay that we employ is complicated and is based on a
depleted nuclear extract. Results obtained can be Obscured by other steps in the
transcription cycle such as elongation, termination, pausing, and recycling. Therefore,
we postulated that a transcription system based on purified components could potentially
be dependent on TFIIF, because TFIIF plays important roles both in initiation,
isomerization, elongation, and recycling. We were particularly interested in how TFIIF
mutants would behave in such an assay. A transcription assay based on purified
components was adapted and developed (Malik et al., 1998; Parvin and Sharp, 1993) .
This system consisted of purified calf thymus RNA polymerase II, recombinant human
TFIIA, TFIIB, TFIIE, TBPc (core TATA Binding Protein), TFIIF, and negatively
supercoiled DNA template. The use Of a negatively supercoiled template obviated the
requirement for the general initiation factor TFIIH (Holstege et al., 1996), which is

necessary for promoter melting using relaxed DNA.

92

Figure 11. TFIIF C-teminal deletion and point mutants in a purified transcription assay
system. Recombinant TBPc, TFIIB, TFIIA, TFIIE, and TFIIF and calf thymus RNA
polymerase II were used in a defined transcription system with supercoiled
pML(C2AT)A71 DNA that contains a G-less cassette downstream from the
transcriptional start site. Both bands (denoted with the arrows) represented specific
transcription products because they were sensitive to Ot-amanitin (lane 1), dependent on
RNAPII and general factors, and resistant to digestion with RNase T1 (data not shown).

TFIIF C-teminal deletion and the point mutants tested do not have any visible

transcriptional defects in this assay.

93

 

Deletion
Mutants

_

TFIIF Point Mutants

 

 

l4

13

12

ll

10

Figure 11

94

The results from the above experiment revealed that TFIIF C-terminal deletion
and point mutants spanning from a.a. 472 to 513 do not have any discemable
transcriptional defects (Figure 11). Transcription in this assay does not depend on RAP74
C-terminal sequence. TFIIF (1-217) mutant, which represents a truncation of 300 amino
acids from the C-terminus, supports the same level Of activity (100%) as native TFIIF
(data not shown). It is possible that RNA polymerase II and some of the other general
transcription factors can compensate for any binding defects between TFIIB and TFIIF.
Alternatively, the nuclear extract system may contain factors that make the interaction

between TFIIB and TFIIF more important for transcription.

Interaction between T F 11F C-terminal Point Mutants and T FIIB

Using BIACORE, TFIIF C-terminal point mutants were measured for their
interaction with TFIIB. 200 nM of each protein was used to determine a qualitative
assessment of the binding (Figure 12 and 13). A summary of the binding results is

presented below in conjunction with multiple round transcription data:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TF 11F Mutant TFIIB Binding Transcription *
LL473AA + 56 % (Moderate Defect)
KK475EE - 69 % (Moderate Defect)
KK480EE - 93 % (N0 defect)

EQT487AAA - 101 % (N0 defect)
VL492AA - 66 %(Moderate Defect)
IL496AA - 78 % (Mild to N0 Defect)
KR498EE - 82 %(Mild to No defect)

LNP500AAA + 60 %(MOderate Defect)
RK504EE - 56 % (Moderate Defect)
M1506AA + 48 % (Defect)

MHF511AAA + 52 % (Defect)

Table 2

* Relative to wild type TFIIF

95

Figure 12. BIACORE qualitative analysis Of TFIIF point mutation complexes capable Of
binding to immobilized TFIIB. 200 nM Of each protein was used to determine a

qualitative assessment of the binding.

96

 

 

 

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97

Figure 13. BIACORE qualitative analysis of TFIIF point mutation complexes
compromised for TFIIB binding. 200 nM of each protein was used to determine a

qualitative assessment Of the binding.

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99

hRAP74 470 KDLLKKFQTKKTGLSSEQTVNVLAQILKRLNPERKMINDKMHFSLKE 517
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492
Indicates TFIIF mutants that are able to bind TFIIB.

It was previously reported that TFIIF mutants 1-450, 1-472, and 1-492 are unable
to bind TFIIB. Based on those studies, we defined the region between amino acid 492
and 517 that was critical for TFIIB interaction. The BIACORE studies with the TFIIF
point mutants further indicates that the region between 475 and 505 is important for
TFIIB interaction. Surprisingly, the LNP500AAA substitution does not affect TFIIF-
TFIIB interaction despite the drastic amino acid substitution; however reversing the
charge on the residues adjacent to those amino acids disrupts TFIIF-TFIIB interaction.
Interestingly, the defects in TFIIBzTFIIF interaction did not correlate with any significant
transcriptional impairment, as evidenced by multiple and single round transcription in

Figure 10.

Expression And Purification Of T FIIB Deletion Mutants.

To systematically localize the region(s) of TFIIB important for TFIIF interactions,
24 TFIIB mutants comprising C-terminal , N- terminal, and short internal deletions were
expressed in and purified to near homogeneity from bacteria (Figures 14 and 15). Using
gel mobility shift assays, these mutants were previously characterized for their ability to
form TBP-TFIIB-DNA complexes (Hisatake et al., 1993) (Figure 14). We obtained these
mutants to study the domains of TFIIB important for TFIIF interaction. These TFIIB
mutants had certain solubility and expression problems that made them challenging to use

in BIACORE. To compensate for these difficulties, we instead used gel mobility shift.

100

 

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Figure 14. Effects of N- and C-terminal deletions of TFIIB on DB complex formation
and basal transcription. Summary of mutant TFIIB proteins obtained from M. Horikoshi.
Figure was adapted from published Nature 363:744-747 paper. Inner two right columns
summarize previously published gel mobility shift data of DB complexes and basal
transcription functional analyses. Outer two right columns summarize data obtained from
our gel mobility shift data of DBPolF complexes and basal transcription functional

analyses (see text for more details). Key: nt - not tested; Basal Txn — basal transcription.

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102

Figure 15. SDS-PAGE of TFIIB mutants stained with Coomassie blue. Panel A) and
Panel B) represent TFIIB internal deletion mutants. Panel C) represents TFIIB N- and C-
terminal truncation mutants. Histidine-tagged recombinant TFIIB were overexpressed in
E. coli BL21(DE3) ArgU and purified to near homogeneity from inclusion bodies by
affinity chromatography on Ni2+-NTA resin. Key: M) Protein standard marker; wt) wild

type TFIIB.

103

 

   

 

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assays to look at the recruitment of RNA polymerase II and TFIIF to the TBP-TFIIB-
DNA complexes. The hypothesis is that if TFIIF:TFIIB interaction is compromised or
completely abrogated, RNA Polymerase II and TFIIF will not be recruited to TBP-
TFIIB-DNA preinitiation complex. It has long been speculated that the N-terminus of
TFIIB is required for RN APII recruitment (via TFIIF) to the preinitiation complex. It has
previously been shown by gel mobility shift and basal transcription assays that disruption
of the N-terminal domain of TFIIB, and in particular the Zinc finger, eliminates

transcriptional activity and recruitment of RNAPII (Barberis et al., 1993; Buratowski and

Zhou, 1993).

T FIIB N— Terminal Deletions Support Recruitment 0f RNA Polymerase II.'TFIIF Wt Into

The Preinitiation Complex.

Using an electrophoretic mobility shift assay, N-terrninal deletions of TFIIB A4-
11, A6-20, A3-3 7, A3-55, and A7-69, were shown to be capable of supporting active
recruitment of RN APllzTF IIF into the preinitiation complex apparently with the same
affinity as wild type TFIIB (Figure 16, compare lanes 5-9 with lane 4). TFIIB mutants
A3-86 and A3—103 were slightly impaired for the formation of the DBPolF complex
(Figure 16, lane 11). The results indicate that the N-terminal domain can be removed up
to the first C-terminal direct repeat of TFIIB and still maintain recruitment of RNAPII
and TFIIF. In this same assay, C-terminal deletions of TFIIB A244-3l6, A279-316, and
A305-316 can support weak recruitment of the DBPolF complex, with TFIIB A244-
316, A279-316 being slightly more disabled (Figure 16, lane 12-15) and collapsing into

an unidentified complex (Figure 16, asterisk).

105

A gel mobility shift assay with the short TFIIB internal deletions corroborated the
results above (Figure 17). Mutants removing segments of the N-terminus are able to
recruit RN APllzTFIIF into the preinitiation complex. TFIIB internal deletions Al 1-

24, A27-44, A45-64, A67-80, A83-103 formed distinct DBPolF complexes (Figure 17,
lanes 5-9). TFIIB internal deletion mutants within the C-terminal domain (A106-

121, A127-144, A148-l63, A163-183, A187-206, A208-227, A226-246, A249-

269, A270—287) formed very unstable DBPolF complexes, also collapsing into an
unidentified complex (Figure 17, lanes 10-18 asterisk). These mutants previously had
been shown to be compromised for DB complex formation (Figure 14) (Hisatake et al.,
1993), suggesting that the ability to form a stable DBPolF does not require TFIIF:TFIIB
but rather requires a stable DB preinitiation complex. Taken together, these results

suggest that no specific sequence between a.a. 11-103 is required for DBPolF assembly.

T FIIB N-Terminal Deletions Support Recruitment 0f RNA Polymerase II: TFIIF(1-217)
Into The Preinitiation Complex.

Previously, we showed that the C-terminus region of RAP74 spanning amino
acids 492 to 517 is critical for TFIIF :TFIIB interaction. TFIIF complex mutants
containing RAP74(1-217) are not able to bind TFIIB as shown by BIACORE analysis
(data not shown). TFIIF (1 -217) was used to test the importance of the C-terminus of
RAP74 for the recruitment of RNAPllzTFIIF to the preinitiation complex with TFIIB
mutants. Gel mobility shift assays with TFIIF(l-217) and TFIIB mutants mirrored the
results obtained with wild type TFIIF (Figures 18 and 19). TFIIB N-terminal truncation

and internal deletion mutants are fully able to recruit Polymerase:TFIIF(1-217) into the

106

DB complex (Figure 18, lanes 5-11 and Figure 19, lanes 5-9). Truncation mutants of
TFIIF (1-492, 1-172, 1-158) were also capable of being recruited with RNAPII to
preinitiation complexes containing wild type and TFIIB N-terminal mutants (data not
shown).

C-terminal TFIIB deletion mutants are very unstable for the formation of DBPolF
complexes containing truncation mutants of TFIIF (1-492, 1-217, 1-172, 1-158) (Figure
20, lanes 5-9). These TFIIB mutants had previously been shown to be incapacitated for
DB complex formation, presumably because the mutations disrupt the direct repeat
domains responsible for interacting with promoter bound TBP (Figure 14). Adding TFIIE
can compensate for TFIIB mutants that do not have the capacity to stably recruit
RNAPllzTFIIF . A representative gel mobility shift experiment with TFIIB(A208-227)
showed that TFIIE allows the formation of DBPolFE complexes containing C-terminal
truncated versions of TFIIF (Figure 20, lanes 10-14). TFIIE may work to stabilize the
preinitiation complex (DB) allowing polymerase to dock.

The collective gel mobility data seems to indicate that TFIIF:TFIIB interaction
through the C-terminus of RAP74 may not be important for the recruitment of RNAPII to
the preinitiation complex in vitro. While TFIIF is absolutely required for the delivery of
RN APII to the preinitiation complex, that delivery does not appear to be mediated
through TFIIBzTFIIF interaction, or interaction between TFIIB-N and RNAPII. It is
possible that polymerase may have undescribed pathways for docking to the preinitiation

complex .

107

Figure 16. Gel mobility shift assay of TFIIB N-terminal and C-terminal deletion
mutants. DBPol complex formation is inefficient without TFIIF (lanes 3-4). N-terminal
deletions of TFIIB A4-11, A6-20, A3-3 7, A3-55, A7-69, were all able to support active
recruitment of RNAPllzTFIIF into the preinitiation complex apparently with the same
affinity as wild type TFIIB (lanes 4-8). TFIIB mutants A3-86 and A3—103 were slightly
unstable for the formation of DBPolF complex. C-terminal deletions of TFIIB A244-
316, A279-3l6, and A305-3l6 can support weak recruitment of the DBPolF complex,

with TFIIB A244-3l6, A279-316 being slightly more disabled (lanes 12-15) and

collapsing into an unidentified complex (asterisk).

108

 

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109

 

Figure 17. Gel mobility shift assay of TFIIB internal deletion mutants. TFIIB internal
deletions Al 1-24, A27-44, A45-64, A67-80, A83-103 formed distinct DBPolF complexes
just as wild type DBPolF complexes (lanes 4-9). Internal deletions within the C-terminal
direct repeats of the protein do not form stable DBPolF complexes (lanes 10-18) and

collapse into an unidentified complex (asterisk).

110

 

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111

 

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Figure 18. Gel mobility shift assay of TFIIB N-terminal, C-terminal deletion mutants
using TFIIF (1-217). TFIIB N-terminal truncation mutants are able to recruit
RNAPII:TFIIF(1-217) into the DB complex (lanes 5-11). Similar to result using wt
TFIIF, TFIIB mutants A3-86 and A3—103 were slightly impaired for the formation of the
DBPolF complex (lanes 10-11), while C-terminal deletions of TFIIB A244-316, A279-

316, and A305-316 support very poor recruitment of the DBPolF complex (lanes 12-14).

112

 

 

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113

Figure 19. Gel mobility shift assay of TFIIB internal deletion mutants using TFIIF (1 -
217). Similar to experiment done with wild type TFIIF, TFIIB N-terminal internal
deletion mutants A1 1-24, A27-44, A45-64, A67-80, A83-103 formed stable DBPolF

complexes (lanes 4-9). Internal deletions at the C-terminal domain do not form stable

DBPolF complexes (lanes 10-18).

114

 

 

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115

 

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Figure 20. Compensation of the defective TFIIB internal deletion A208—227 with TFIIE
as seen by gel mobility shift assay. TFIIB internal deletion A208-227 is not capable of

stably recruiting RNAPII: TFIIF complexes (wt, l-492,1-217,1-172,1-158). The addition

of TFIIE stabilizes formation of the DBPolFE complex (lanes 10-14).

116

 

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117

Reconstituted Transcription Assay With Purified Components And TFIIB Mutants.

Using a reconstituted transcription assay with recombinant human TBPc, TFIIA,
TFIIB, TFIIE, TFIIF, and a negatively supercoiled DNA template (Malik et al., 1998;
Parvin and Sharp, 1993) the ability of the TFIIB mutants was assessed to support basal
transcription. These mutants had previously been assayed for basal transcription, albeit in
a cruder assay system using partially purified components such as TFIID/TBP and
TFIIE/TFIIF fraction, RNAPII and recombinant TFIIB (Hisatake et al., 1993). Although,
the N-terminal domain was dispensable for DB complex formation; in the transcription
assay, disruption within the N-terminal Zinc finger domain up to the first direct repeat
eliminated or markedly reduced transcription. Residues in this region whose removal
reduced, but did not eliminate transcription included amino acids 6-20, 67-80, and 83-103
(Figure 14). All mutations that eliminated DB-promoter DNA complex formation (such
as C-terminal disruptions) also eliminated basal transcription. In our revised basal
transcription assay, total disruption of TFIIB’s N-terminus (Figure 21, lanes 13-17) and
specific deletion of the Zinc finger (A1 1-24, A27-44) eliminated transcription (Figure 21,
lanes 4-5). In contrast to TFIIB A1 1-24 and A27-44, but similar to previous results, N-
terminal deletion A6-20 (Figure 21, compare lane 12 to lanes 4-5) reduced but did not
abrogate transcription. Regions within the N-terminus (A67-80, and A83-103) whose
deletion did not eliminate transcription were also observed as previously characterized
(Figure 21, lanes 7-8). Disruptions flanking both these regions (A45-64 and A106-121),
however, indicated that our assay is slightly more sensitive than the one previously used,
as deletions within those regions were also capable of supporting low level basal

transcription. Though the TFIIB AlO6-121 was severely incapacitated for basal

118

transcription (Figure 21, lane 9). Mutations that eliminated DB complex formation by
interfering with TFIIB’s direct repeats (A187-206 and A208-227) also eliminated basal
transcription (Figure 21, lanes 10-11). The above functional data indicate that the region
between a.a. 11 to 45 is required for basal transcription. Because all the TFIIB mutants
were purified under denaturing conditions, the presence of guanidine hydrochloride could
have obscured the obtained results. However, as a control, TFIIB purified under native
conditions was used to assess the effect of guanidine in this sensitive assay. TFIIB
purified under denaturing conditions supported the same level of basal transcription as

TFIIB purified under native conditions (Figure 21 , compare lane 3 to 2).

119

Figure 21. TFIIB N- and C-terminal mutants in a purified transcription assay system.
This assay is similar to the one described in Figure 11. Disruption of TFIIB’s N-terminus
(lanes 13-17) and specific deletion of the Zinc finger (A1 1-24, A27-44) eliminated
transcription (lanes 4-5). Mutations compromising DB complex formation by interfering
with TFIIB’s direct repeats (A187-206 and A208-227) also eliminated basal transcription
(lanes 10-1 1). N-terminal deletion A6-20 (lane 12) reduced but did not abrogate
transcription. Regions within the N-terminus (A67-80, and [183-103) whose deletion did
not eliminate transcription were also observed (lanes 7-8). In contrast to results published
elsewhere, disruptions flanking both these regions (1145-64 and A106-121) (lanes 6 and 9)
were also capable of supporting low level basal transcription. Although TFIIB A106-121
is significantly more impaired than TFIIB A45-64. Lane 1) a-Amanitin added to the

complete reaction inhibiting RNAPII transcription. Lane 2) Complete reaction containing

recombinant TBPc, TFIIB (not in guanidine HCl), TFIIA, TFIIE, wt TFIIF, and
supercoiled pML(C2AT)A71 DNA . Lane 3) Identical to Lane 2 but with TFIIB diluted

from guanidine HCl stock. Lanes 4-17) TFIIB mutants.

120

 

 

 

 

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121

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DISCUSSION

Evidence suggests a functional interaction between TFIIF and TFIIB. These
factors cooperate to bring RNAPII into the preinitiation complex (Buratowski et al.,
1989; Conaway et al., 1991; Serizawa et al., 1994; Zawel and Reinberg, 1993).
Furthermore, in vivo studies with yeast have established a genetic relationship between
the RAP74 subunit of TFIIF and TFIIB that can affect transcription initiation start-site
selection (Sun and Hampsey, 1995).

TFIIB contains at its N-terminus a Zinc finger domain that is dispensable for
interaction with TBP, but is important for efficient recruitment of RNAPII-TFIIF, support
of basal transcription initiation (Barberis et al., 1993; Buratowski and Zhou, 1993;
Hisatake et al., 1993) and accurate start-site selection in vivo (Pardee et al., 1998; Pinto et
al., 1994). Mutations at the N-terminus inhibit assembly of transcription intermediates
presumably because they fail to interact with either TFIIF or RNAPII (Buratowski and
Zhou, 1993; Ha et al., 1993; Hisatake et al., 1993; Malik et al., 1993). The N-terminal
domain of TFIIB binds the RAP30 subunit of TFIIF and RNA polymerase II (Ha et al.,
1993; Hisatake et al., 1993). At the C-terminus of TFIIB lies two imperfect direct repeats
similar to the core domain of cell cycle regulatory proteins (Bagby et al., 1995; Gibson et
al., 1994). The two cyclin-like C-terminal repeats of TFIIB (TFIIBc) are necessary and
sufficient for interaction with the TBP-TATA complex (Hisatake et al., 1993). Based on
the three-dimensional structure of TFIIBc (Bagby et al., 1995) and the TFIIBc-TBP-DNA

ternary complex (N ikolov et al., 1995), the C-terminal region of TFIIB binds TBP,

122

activators (Roberts and Green, 1994) and DNA. The N-terminus is postulated to be a
scaffold for the assembly of RNA polymerase II-TFIIF.

In addition to the functional interaction between TFIIB and TFIIF during
transcription initiation, both factors may control the dephosphorylation of the Carboxy
Terminal Domain of RNA polymerase 11 after transcription termination (Chambers et al.,
1995) in a process known as recycling. Dephosphorylated RNAPII preferentially enters
the preinitiation complex, and CTD kinases phosphorylate RNAPII within the
preinitiation complex or shortly after initiation (Chambers and Dahmus, 1994; Laybourn
and Dahmus, 1989). During elongation, RNAPII is hyperphosphorylated on the CTD
(Chambers and Dahmus, 1994; Lu et al., 1991). The CTD phosphatase has been
independently isolated by HeLa cell extract fractionation (Chambers and Dahmus, 1994)
and a two-hybrid screen for RAP74 C-terminal interacting proteins (Archambault et al.,
1997), hence its name FCPl (TFIIE-associating QT D phosphatase). Despite its role in
CTD dephosphorylation, Fcplp does not resemble known eukaryotic protein
phosphatases, and it is essential for most transcription by RNAPII in vivo (Kobor et al.,
1999). The C-terminal domain of RAP74 stimulates a CTD phosphatase (Archambault et
al., 1997; Archambault et al., 1998; Chambers et al., 1995). The C-terminal domain of
RAP74 is also responsible for interaction with TFIIB (Fang and Burton, 1996) and
RNAP 11 (Wang and Burton, 1995). TFIIB which can directly bind the CTD phosphatase
(Jack Greenblatt, unpublished data), blocks stimulation of CTD phosphatase activity by
RAP74 (Chambers et al., 1995). Because RAP74 stimulates and TFIIB blocks

stimulation of CTD phosphatase activity (Chambers et al., 1995), TFIIB has been

123

suggested to be present in elongation complexes to block CTD dephosphorylation in
order to prevent premature termination (Lei et al., 1998).

Multiple-round transcription in vitro using a Hela extract system appears to reflect
a physiological recycling system, because this process involves activation of transcription
complexes as a function of time (Lei et al., 1998). We originally initiated RAP74 C-
terminal truncation mutagenesis to study the effects of that region in initiation and
multiple round transcription. Three RAP74 C-terminal mutants (1-492, 1-472, and 1-
450) were constructed and reconstituted with RAP30 to make TFIIF. All the RAP74 C-
terminal truncation mutants were severely defective in the multiple-round transcription
assay . In particular, RAP74(1-492) mutant, which represented a truncation of 25 amino
acids, was significantly compromised for transcription . This truncation reduced the total
transcription signal by more than 50 % (Figure 3B). Using biosensor surface plasmon
resonance studies (BIACORE), we determined that these RAP74 C-terminal truncation
mutants when complexed with RAP30 were defective for their interaction with the
general initiation factor TFIIB (Figure 8). Similar to wild type RAP74, the RAP74 C-
terminal truncation mutants by themselves were able to stably associate with TFIIB. It is
possible that a conformational change occurs in RAP74 as a result of its association with
RAP30 that modifies the C-terminus for TFIIB binding . This conformational change
could enhance the overall affinity of TFIIF for TFIIB (3 to 10X higher affinity) as
opposed to RAP74 or RAP30 interaction with TFIIB by themselves (Figures 5 and 4).
Because TFIIF and TFIIB are intimately intertwined at the most crucial stages of the
transcription cycle, we speculated that defects in transcription as seen with the C-terminal

RAP74 mutants could be due to compromised binding with TFIIB.

124

As expected, both TFIIF subunits were capable of binding TFIIB individually,
with tight binding affinities (Figure 4). TFIIF is thought to mediate the interaction
between RNAPII and the DB complex and this function had long been attributed
specifically to the RAP30 subunit (Flores et al., 1991). Consistent with this premise,
TFIIB binds to RAP30, and this interaction has been mapped to the N-terminal domain of
TFIIB (Fang and Burton, 1996; Ha et al., 1993). However, second site suppression
genetic experiments in yeast have implicated RAP74 interaction with TFIIB (Sun and
Hampsey, 1995). Both subunits of TFIIF, therefore are possibly involved in TFIIB
binding. However, the N-terminus of RAP74 has been shown to block RAP30:TFIIB
interaction by binding the N-terminal region of RAP30 (Fang and Burton, 1996).
RAP30’s TFIIB interaction domain overlaps its RAP74 binding domain. Also, by
sequestering TFIIB, the C-terminus of RAP74 can compete for TFIIB binding and
dissociate RAP30 :TFIIB interactions (Fang and Burton, 1996). This indicates that when
TFIIF is intact, TFIIBzTFIIF contact would be maintained through the C-terminus of
RAP74 and the N-terminus of TFIIB. If RAP30:TFIIB interaction is physiologically
important, the TFIIF complex would have to dissociate within some complexes.

The TFIIF:TFIIB binding dissociation constant (K0) was 1.43 x 10'3 M,
indicating a strong binding interaction (Figure 5). The TFIIF:TFIIB-N binding showed
slightly higher affinity than TFIIF:TFIIB with a KD of 1.25 x 10'8 M ( Figure 7). Roger
Kornberg’s lab has attempted to observe interactions between yeast transcription factors
using a similar biosensor system, but has not been able to detect the TFIIF :TFIIB
interaction (Bushnell et al., 1996). However, the concentration and purity of yeast TFIIF

that was used was low and consequently the detection was near the low limit of the assay.

125

BIACORE binding studies with TFIIB and the adenovirus ElA 13 S transcriptional
activator (Paal et al., 1997) have indicated that the dissociation constant is within the
same order of magnitude (10’8 M) as the value that has been calculated for TFIIF:TFIIB
and TFIIF:TFIIB-N. The C-terminal repeats of TFIIB do not appear to interact with
TFIIF, based on the flat sensorgram for the interaction (Figure 7). The solved 3D
structure of the TATA -TBP-TFIIBc complex strongly indicated that the interaction
between TFIIB and TFIIF might be facilitated by the N-terminal region of TFIIB

(N ikolov et al., 1995). The TFIIF:TFIIB-N BIACORE analysis corroborates the inference
previously made based on the crystallographic structure of TFIIB-C (Figure 7).

Based on multiple round transcription and BIACORE analysis, RAP74 sequence
from 492 to 517 was deemed important for multiple round transcription and binding to
TFIIB when RAP74 was complexed with RAP30. Point mutations in a.a. 470 to 517
region of full length RAP74 were made to identify regions of the C-terminal domain that
are important for transcriptional function and TFIIB interaction. The BIACORE studies
with the TFIIF point mutants indicated that the region between RAP74 a.a. 475 and 505
was critical for TFIIB interaction (Figures 12 and 13). Interestingly, the LNP500AAA
substitution did not affect TFIIF-TFIIB interaction; however reversing the charge on the
residues adjacent to those amino acids disrupted TFIIF-TFIIB interaction. Noticeably,
charged residues K476, K480, K481, K498, K499, R504, K505 are conserved between
human, Drosophila, and yeast RAP74 (Figure 9). It is possible that these residues may
form a salt bridge with acidic amino acids on TFIIB and thereby form the basis for the
TFIIF-TFIIB interaction. The N-terminus of TFIIB harbors several acidic amino acids

that are conserved between human, Drosophila, and yeast TFIIB (data not shown). We

126

know from BIACORE data that TFIIF binds slightly more strongly to the N-terminus of
TFIIB than to the full-length protein. It is plausible that the reason why TFIIF binds
slightly better to the N-terminus of TFIIB is because the conserved acidic amino acid
groups are unmasked as a result of the truncation of the C-terminus, allowing TFIIF easy
access to the TFIIB region responsible for that interaction.

Transcriptional defects seen in multiple round transcription appear to be more
complicated than simple TFIIBzTFIIF interaction disruptions. Using the RAP74 C-
terminal point mutants, there did not seem to be a clear linkage between transcription
function and TFIIBzTFIIF interaction (Figures 10, 12, 13, Table 2). Although, the TFIIF
C-terminal point mutants described have some transcriptional defects, these cannot be
attributed totally to compromised TFIIBzTFIIF, because some of the same C-terminal
point mutant (TFIIF: LL473AA, LNP500AAA, MISO6AA, MHF51 lAAA) could still
maintain TFIIBzTFIIF interaction.

The above results may implicate the RAP74 C-terminus in CTD phosphatase
interaction and stimulation rather than TFIIB interaction. Transcriptional defects may be
due to disruptions in the functional complex responsible for polymerase recycling and
CTD phosphorylation /dephosphorylation. Changes in the affinity of TFIIF for CTD
phosphatase could affect elongation and polymerase’s ability to reinitiate. The calf
thymus RN APII used in the reconstituted purified components transcription was prepared
by the method of Hodo and Blatti (Hodo and Blatti, 1977) and was primarily in the IIb
form, lacking the carboxy terminal domain (CTD). Moreover, CTD kinases and
phosphatases are not included in the purified components transcription system. Therefore,

CTD phosphorylation and dephosphorylation are not relevant in the reconstituted

127

transcription system. This might explain why no transcriptional defect is seen with TFIIF
C-terminal truncation (including TFIIF(1-217)) and point mutants in the purified
transcription assay (Figure 11). TFIIF C-terminal mutations affecting CTD phosphatase
interaction may, therefore, be the main reason for the presumed inhibition of RNAPII
recycling in the HeLa TFIIF-depleted nuclear extract system .

Evidence suggests that TFIIB is conforrnationally pliable and that TFIIB N- and
C- terminal regions are involved in an intramolecular interaction that potentially
sequesters the N- terminus from associating with other factors (Roberts and Green, 1994).
If TFIIF interacts with TFIIB’s N terminal domain as a docking scaffold, then TFIIB
must be conforrnationally unlocked where the N and C terminal intramolecular
interactions are disrupted in order to recruit TFIIF interaction. This disruption can be
facilitated by specific activators such as VP16 (Roberts and Green, 1994). This model is
consistent with recent structural studies of human TFIIB showing that interaction of either
VP16 or the N-terminus of TFIIB with the C-terminus of TFIIB induce distinct changes
in the orientation of the two repeat domains of the C-terminal region of TFIIB relative to
each other (Hayashi et al., 1998). The studies however did not address the potential role
of TFIIB conformational changes on transcription. Recently, an activation-specific role
for TFIIB in vivo was observed with an activator-induced TFIIB conformational change
that may facilitate PIC assembly (Wu and Hampsey, 1999). The yeast activator Pho4’s
interaction with TFIIB induces a conformational change that might represent disruption of
the nIIB-cIIB interaction which then stimulates PH05 transcription in vivo. Some
activators may stimulate transcription by inducing a conformational change in TFIIB that

drives preinitiation complex assembly forward.

128

We obtained C-terminal , N- terminal, and short internal deletion TFIIB mutants
to localize regions of TFIIB important for TFIIF interactions (Hisatake et al., 1993)
(Figure 14). These TFIIB mutants had certain solubility and expression problems which
made them challenging to use in BIACORE. To compensate for these difficulties, we
instead used gel mobility shift assays to look at the recruitment of RNA polymerase II
and TFIIF to the TBP-TFIIB-DNA complexes.

The results of the gel mobility shift experiments indicated that removal of the N-
terminal domain of TFIIB up to the first direct repeat of the protein was able to maintain
recruitment of polymerase and TFIIF. TFIIB sequence from amino acid 11 to 103 is
dispensable for the assembly of DBPolF complexes (Figure 16-19) but amino acid
sequence between 11 to 45 is required for basal transcription (Figure 21). These results
were in marked contrast to those previously published in which N-terminal disruptions
that affected the Zinc finger domain could form a DB complex but could not recruit PolF
to form DBPolF (Buratowski and Zhou, 1993). The reagents used previously were Hela
fractionated components that might have harbored contaminants which could have
obscured these results. In our assay we have used recombinant protein for all the general
transcription factors and purified calf thymus RNAPII. Functional data indicated that the
region between a.a. 11 to 45 is required for basal transcription. This region encompasses
the Zinc finger domain, located between a.a. 15 to 37, and suggests that the region
immediately flanking the Zinc finger is also important. Moreover, partial deletion of the
Zinc binding domain (A6-20) severely compromises basal transcription but does not
eliminate it. Deletion of region 45-103 can maintain moderate level of transcription,

whereas further deletion beyond this region (affecting DB complex formation) does not

129

support basal transcription as expected. The Zinc finger and the region immediately
flanking it are essential for basal transcription but are not required for recruitment of
RNAPII and TFIIF (Figure 21).

It has long been thought that TFIIB’s Zinc finger could recruit RNAPII through
protein-protein interaction with TFIIF and/or polymerase. Zinc finger motifs can function
as a metal-linked interaction domain. In fact, several RNA polymerase II subunits contain
zinc-binding domains that are known to be functionally important (Treich et al., 1991).
Our data show that the N-terminal region of TFIIB is not primarily required for the
recruitment of polymerase and TFIIF but rather for another function in initiation. This
finding does not diminish possible TFIIB:TFIIF interactions important downstream of the
pathway, though recruitment of polymerase to the PIC may take place through
undescribed docking interactions with the DB complex. TFIIB mutations that disrupt the
C-terminal repeats form very unstable DBPolF complexes that collapse into a complex
which, based on size, might be a DBF complex having lost polymerase (Figures 16 and
17, asterisk). These C-terminal mutants have been shown to be compromised for DB
complex formation (Figure 14) (Hisatake et al., 1993). The inability to form a stable DB
complex, a result of disruption of TFIIB domains that interact with TBPzDNA, is the
cause for poor recruitment of RNAPII:TFIIF. The addition of TFIIE can partially
compensate for TFIIB’s inability to form a stable DB and recruit RNAPII:TFIIF to form
a DBPolFE complex (Figure 20) but does not restore function (Figure 21). TFIIE

contributes to DNA wrapping around RNA polymerase within the PIC (Robert et al.,

1998; Robert et al., 1996) which may compensate for defective TBPzDNAzTFIIB

complexes. TFIIE-0t has a zinc-ribbon motif and yeast TFIIE binds single-stranded DNA,

130

which suggests that TFIIE may participate to form or stabilize the melted DNA in the

initiator region (Kuldell and Buratowski, 1997).

131

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