.1.
.t 334
k. wk. ;
i... .Lz...

3..
i .
in
.2.
. ..
arena.
31: .n.«.
a . z.
4....an ..
tuft... :
u...33

1.. \u a:

x l ,
A... I <

1|. 5:: 32} 1 .

31.2.... n1.) 4

. t .444“...

5:...“ 3 in?

2.3.1.. ; ‘
.. r. ,
‘wflxluwihmky

: . , . 3E?

E awfiwiiéauéi

in 4.3% a .

 

ll Illllflllllllllllllllllllllfllll

3 1293 01410 2465

 

This is to certify that the

dissertation entitled

The Structure and Function of Human RAP30, the Small
Subunit of General Transcription Factor TFIIF

presented by

Shi—Min Fang

has been accepted towards fulfillment
of the requirements for

Ph . D degree in Biochemistry

 

 

 

Date 114/96

 

M5 U is an Affirmative Action/Equal Opportunity Institution 0- 12771

 

LIBRARY
Michigan State
University

 

 

 

rues a serum BOX to remove this checkout train your record.
TO AVOID FINES return on or before dd. duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

usu IIAn Affirmative Action/Emmi OW imwon
Wan-M

THE STRUCTURE AND FUNCTION OF HUMAN RAP30, -
THE SMALL SUBUNIT OF GENERAL TRANSCRIPTION FACTOR TFIIF

BY

Shi-Min Fang

A DISSERTATION

Sumitted to
Michigan State University
in partial fulfilment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Biochemistry

1995

ABSTRACT

THE STRUCTURE AND FUNCTION OF HUMAN RAP30,
THE SMALL SUBUNIT OF GENERAL TRANSCRIPTION FACTOR TFIIF

BY

Shi-Min Fang

RAP30 is the small subunit of TFIIF (RAP30/74), a general initiation and
elongation factor for transcription by RNA polymerase II. RAP30 has functions
analogous to the functions of bacterial sigma factors. It binds to RNA polymerase H,
recruits RNA polymerase II to the pre-initiation complex, prevents RNA polymerase II
from binding nonspecifically to DNA, and greatly inhibits nonspecific transcription by
RNA polymerase II. Applying a 2-dimensional sequence analysis method called
Hydrophobic Cluster Analysis, we have aligned RAP30 to bacterial sigma factors.
RAP30 contains sequences weakly similar and co—linear with conserved subregions 1.2,
2.1, 3.1, and 4.1 of sigma factors. RAP30 also has sequence similar to the N-terminal
region of delta protein, a non-essential subunit of B. sublilis RNA polymerase.

In order to understand the relationship between the structure and the function of
RAP30, a set of deletion mutants of RAP30 was constructed. Mutants were tested for
accurate transcriptional activity, RAP74 binding, RNA polymerase II binding and TFIIB
binding. Transcription assays indicate the importance of both N- and C-terminal regions
for RAP30 function. RAP74 binds to the N-terminal region of RAP30 between amino
acids 1-98. Two regions of RAP30, one near the N-terminus and one within the central
region, are important for RNA polymerase II binding. Multiple contacts within the

RAP74—binding and RNA polymerase II-binding regions of RAP30 contribute to TFIIB

binding. Deletion of the N-terminal region of RAP30 abolishes RAP74-binding, RNA
polymerase II-binding and TFIIB binding and activates the C-terminal region of RAP30
for DNA binding. The N-terminal region of RAP30, therefore, may be a domain that
regulates the accessibility of cenual and C-terminal domains.

Several lines of evidence indicate that there may be significant coupling of TFIIF
and TFIIB function in transcription. Both RAP30 and RAP74 bind independently to
TFIIB. Analysis of deletion mutants of RAP74 shows that a C-terminal region between
amino acids 358-517 binds directly to TFIIB, and this region of RAP74 also binds to
RNA polymerase 11. Interestingly, RAP74 antagonizes the interaction between TFIIB and
RAP30, both by binding to RAP30 and by binding to TFIIB. RAP30, therefore, binds to
TFIIB only in the absence of RAP74. When the TFIIF complex is intact, TFIIF-TFIIB
contact most likely is maintained through the RAP74 subunit. If these binding
relationships are maintained within functional transcription complexes, dynamic
interactions between TFIIF subunits and TFIIB may be a mechanism to separate RAP30

and RAP74 functions during various stages of the transcription cycle.

To my parents and my homeland

iv

On the River Bank Without A Boat
1 by Shi-Min Fang

flfier so many years,
So many rivers liave dried' up.
‘17iis one cannot last too fimg.

I will'fie a6[e to cross it on foot

To [ho/{for tlie d226ris of paradise.

‘17ie paradise 5uil't wit/i toy Eric/{s
Collapsed'on t/ie otfier sfiore,

find' decayed into tfiose Mick 6&1cfico66les.

’Wlien I arrive at tfie middle of tlie river;
The river will rise quietly.

I will stay at tlie middle qft/ie river,
The river wilZ'rfro'wn me slbwly.

Qfie river is rising quitely at tfiis moment,

flnd'all'qfcolililes are sliining togetfier.

ACKNOWLEDGMENTS

I would like to thank my mentor, Dr. Zachary Burton, for his guidance and
friendship through the years. His contribution to this work has been invaluable. I also
thank the members of my guidance committee, Drs. Jon Kaguni, Lee Kroos, John Linz
and John Wang, for their advice and encouragement.

I acknowledge the supports of the former and present members of Dr. Burton lab,
Dr. Ann Finkelstein, Dr. Wladyslaw Werel, David Chavez, Bo Qing Wang, Chun-hsiang
Chang, Lei Lei, and Stephan Reymez. Special thanks to Ann for teaching me the first
experiment in this lab, to Bo Qing and Chun-hsiang for providing RAP74 proteins and
HeLa cell extracts that were used in this work. The friendship from my colleagues and

friends has made East Lansing such a pleasant place to work and live.

TABLE OF CONTENTS

PAGE
LIST OF TABLES .......................................................................................................... IX
LIST OF FIGURES ........................................................................................................ X
LIST OF ABBREVIATIONS ......................................................................................... XIII
CHAPTER I: INTRODUCTION ............................................................................. 1
Literature Review ................................................................................................ 2
Transcription by RNA Polymerase II ..................................................... 2
Transcription Factors and Promoters .......................................... 2
Template Activation .................................................................... 4
Promoter Recognition and Transcription Initiation .................... 5
Elongation ................................................................................... 6
Termination ................................................................................. 7
Transcription Initiation by RNA Polymerase II ...................................... 8
Multistep Assembly Process ....................................................... 8
Minimal Transcription Machinery .............................................. ll
Holoenzyme Assembly Model .................................................... 12
Transcription Activators ............................................................. l3
Coactivators ................................................................................ 14
RNA Polymerase II ................................................................................. 15
General Transcription Factors ............................. 18
TFIIDCI'BP and TAFs) ................................................................ 18
TFIIA .......................................................................................... 21
TFIIB ........................................................................................... 22
TFIIB ........................................................................................... 24
TFIIH .......................................................................................... 25
TFIIF(RAP30fl4) .................................................................................... 27
Overview ............................................................................................................. 33
References ........................................................................................................... 37
CHAPTER II: SEQUENCE ANALYSIS OF RAP30 .............................................. 49

vii

Abstract ................................................ 50

Introduction ......................................................................................................... 51
Methods ............................................................................................................... 55
Hydrophobic Cluster Analysis ................................................................ 55
Quality of Alignments ............................................................................. 55
Results ................................................................................................................. 57
Human RAP30 is structurally related to bacterial sigma factors ............ 57
The overall structure of yeast Cdc73p is similar to RAP30 .................... 57
Multiple sequence alignment of RAP30 and Cdc73p ............................. 58
Discussion ........................................................................................................... 74
References ........................................................................................................... 77
CHAPTER III: FUNCTIONAL DOMAINS OF RAP30 ........................................ 79
Abstract ............................................................................................................... 80
Introduction ......................................................................................................... 81
Materials and Methods ........................................................................................ 83
Construction of RAP30 mutants ............................................................. 83
In vitro transcription ................................................................................ 84
Gel filtration analysis of RAP30 mutants ............................................... 85
Protein-protein interaction assays ............................................... 85
RAP74 binding to RAP30 mutants ............................................. 85
RNAP II binding to RAP30 mutants ........................................... 85
TFIIB binding to RAP30 mutants ............................................... 86
Results ................................................................................................................. 87
Both N-and C-terminal regions of RAP30 are important for
transcription activity ............................................................................... 87
Modification at C-terminus causes RAP30 to behave as a
monomer .................................................................................. . .............. 87
RAP74 binds to the N—terminal region of RAP30 between amino
acids 1-98 ...... 95
Both N-terminal and central regions of RAP30 are important for
polymerase binding ................................................................................. 95
Multiple contacts within the RAP74-binding and polymerase-
binding regions of RAP30 contribute to TFIIB binding ......................... 100
Discussion ........................................................................................................... 106
References ........................................................................................................... 1 1 1

viii

CHAPTER IV: DYNAMIC INTERACTIONS BETWEEN SUBUNITS OF

TRANSCRIPTION FACTOR TFIIF AND TFIIB .................................................... 113
Abstract ............................................................................................................... 1 14
Introduction ......................................................................................................... 1 15
Materials and Methods ........................................................................................ 119

Protein reagents ....................................................................................... 119
Protein-protein interaction assays ........................................................... 119
TFIIB binding to RAP74 mutants ............................................... 119
ELISA assays .............................................................................. 119
Ni2+-affinity bead procedure ...................................................... 120
Results ................................................................................................................. 121
Both RAP30 and RAP74 bind independently to TFIIB .......................... 121
C-terminal region of RAP74 between amino acids 358-517 binds
directly to TFIIB ..................................................................................... 121
RAP74 antagonizes the interaction between TFIIB and RAP30 ............ 130
RAP74 disrupts the interaction between TFIIB and RAP30 by two
mechanisms ............................................................................................. 135
RAP74-TFIIB interaction is maintained in complexes containing
polymerase .............................................................................................. 135
Discussion ........................................................................................................... 141

References ........................................................................................................... 149

LIST OF TABLES

PAGE
Chapter II
Table 1. HCA alignment scores of hRAP30, dRAP30 and Cdc73p ..................... 69
Chapter 111
Table 1. Molecular weight of RAP30 C-terminal deletion mutants
determined by gel filtration ..................................................................... 94

Figure 1.

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.
Figure 7.

Figure 1.
Figure 2.
Figure 3.
Figure 4.

Figure 5.

LIST OF FIGURES

PAGE
Chapter I
Preinitiation complex formation: the ordered multistep assemble
model and the holoenzyme assemble model ........................................... 9
Chapter II
The N-terminal regions of RAP30 and Cdc73p are similar to
subregion 1.2 of bacterial sigma factors ................................................. 59
RNA polymerase II binding regions of RAP30 and Cdc73p are
similar to the core binding subregion 2.1 of bacterial sigma factors ...... 61
The C-terminal region of RAP30 is similar to subregions 3.1 and
4.1 of bacterial sigma factors ................................................................. 63
The C-terminal regions of RAP30 and Cdc73p are similar to the
N-terminal region of delta protein .......................................................... 65
HCA lots of human RAP30, Drosophila RAP30 and S. cerevisiae
Cdc7 p ................................................................................................... 67
Tire structure of human RAP30 and Cdc73p .......................................... 70
Multiple sequence alignment of human RAP30, Drosophila ’
RAP30, S. cerevisiae ng2p, and Cdc73p, based on HCA .................... 72
Chapter III _
RAP30 deletion mutants ......................................................................... 88
Transcriptional activities of RAP30 deletion mutants ............................ 90
C-terminal modification effects RAP30 activity .................................... 92
Sequences between amino acids 1-98 of RAP30 are sufficient
for RAP74 binding ................................................................................. 96
RAP30 sequences between 1-50, 131-159 and 152-176 are
important for RNAP II binding ............................................................... 98

xi

Figure 6.

Figure 7.

Figure 8.

Figure 1.

Figure 2.
Figure 3.

Figure 4.
Figure 5.
Figure 6.

Figure 7.

Figure 8.

Figure 9.

RAP30 sequences between amino acids 1-176 are required for

tight binding to TFHB ............................................................................. 101
A minimal TFIIB-binding region of RAP30 maps between

amino acids 27-118 ................................................................................. 103
Functional domains of RAP30 ............................................................... 107

Chapter IV

RAP30 and RAP74 deletion mutants ..................................................... 122
Both RAP30 and RAP74 bind independently to TFIIB ......................... 124
RAP74 has a masked binding site for TFIIB located within a C-

terrninal region between amino acids 358-517 ...................................... 126
C-terminal deletion mutants of RAP74 do not bind TFIIB tightly ......... 128
Dynamic interactions between TFIIF subunits and TFIIB ..................... 131

A) RAP74 blocks formation of a RAP30-TFIIB complex
B) TFIIB does not block formation of a RAP30/'74 complex ................ 133

RAP74 blocks formation of the RAP30-TFIIB complex by
binding to RAP30 and by binding to TFIIB .......................................... 136

RNA polymerase II does not appear to block TFIIB-RAP74
interaction ............................................................................................... 138

Models for dynamic TFIIF-TFIIB interactions during initiation
promoter escape, and elongation .......................... . ................................. 145

xii

a.a.
AdMLP
ATP

BSA

Da
DNA
D'IT
EDTA

EGTA
ELISA
GTP
HCA
I-IEPES

nt

NTP

O.D. (490nm)
PAGE

PCR

RAP

LIST OF ABBREVIATIONS
amino acid(s)
adenovirus major late promoter
adenosine triphosphate
base pairs
bovine serum albumin
cytosine triphosphate
Dalton
deoxyribonucleic acid
dithiothreitol
ethylenediamine tetraacetic acid
ethyleneglyCol-bis-(B-aminoethyl ether) N,N,N',N'-tetraacetic acid
enzyme-linked immunosorbent assay
guanine triphosphate
hydrophobic cluster analysis
N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid)
is0propyl- B—D-thiolgalactopyranoside
kilobase pairs
kilodaltons
messenger RNA
nucleotides
nucleoside triphosphate
optical density at 490nm
polyacrylamide gel electrophoresis
polymerase chain reaction

RNA polymerase II associating proteins

xiii

RNA ribonucleic acid

RNAP II RNA polymerase II

rRNA ribosomal RNA

SDS sodium dodecyl sulfate

TAF TBP associated factor

TBP TATA box-binding protein

TCA trichloroacetic acid

TFII transcription factor of RNA polymerase II
tRNA transfer RNA

Single letter abbreviations for the amino acids: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe;
G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T,
Thr, V, Val; W, Trp; and Y, Tyr.

xiv

CHAPTER I
INTRODUCTION

LITERATURE REVIEW

One of the most important ways to modulate gene expression in biological
systems is to regulate transcription. The mechanism of gene transcription in eukaryotes is
more complex and less well understood than in prokaryotes. In prokaryotes a single form
of RNA polymerase, composed of three distinct subunits (0288'), performs all RNA
synthesis. Selective recognition of promoters is determined by a a initiation factor that
associates with the azflfi' "core". By contrast, there are three distinct nuclear RNA
polymerases in eukaryotes. These three polymerases, each containing from 8 to 14
subunits, are responsible for transcribing different sets of genes: RNA polymerase I
synthesizes ribosomal RNA precursors, RNA polymerase II transcribes protein-coding or
class 11 genes, and RNA polymerase III synthesizes SS ribosomal RNA and transfer
RNA. Each of these enzymes requires a variety of auxiliary factors, known» as
”transcription factors", for selective promoter recognition and regulated transcription
initiation, and cannot specifically initiate transcription on its own. In this review, only the

synthesis of messenger RNA by RNA polymerase II is discussed.

Transcription by RNA Polymerase II

Transcription by RNA polymerase II (RN AP II) in eukaryotes involves multiple
steps including: 1) template activation; 2) promoter recognition; 3) transcription
initiation; 4) transcription elongation and 5) transcription termination. Each step requires

a variety of transcription factors and DNA elements.

Transcription Factors and Promoters

Transcription is regulated by the interaction of specific proteins (nuns-factors)
with control signals in the DNA (cis-elements). The trans-factors that regulate
transcription initiation of mRNA can be divided into three classes (Matsui et a1. 1980;
Davidson et al., 1983; Samuels et al., 1982; reviewed by Drapkin et al, 1993). The first
class, termed general transcription factors (GTFs) or basal transcription factors, are
required for basal level transcription initiation on almost all class II promoters (reviewed
by Weinmann, 1992; Zawel and Reinberg, 1993; Conaway and Conaway, 1993;
Buratowaki, 1994; ‘Maldonado and Reinberg, 1995). The second class are sequence-
specific transcription factors including activators and repressors, which regulate the rate
of transcription initiation by binding to the specific DNA elements. They interact with
basal transcription factors directly or indirectly (reviewed by Mitchell and Tjian, 1989;
Ham et al., 1992; Tjian and Maniatis, 1994). The third class consists of cofactors,
mediators or adaptors, which mediate the interactions between activators or repressors
and basal factors to relay regulatory signals to RNAP II. Cofactors can also stimulate or
repress transcription by themselves (Merino et al., 1993; Auble and Hahn, 1993; Ge and
Roeder, 1994; Kretzschmar et al., 1994; reviewed by Zawel and Reinberg, 1995).

The promoter acts as a DNA signal that directs transcription factors and RNAP II
to the initiation site (W eil et al., 1979; Manley et al., 1980). Cis-elements in the
promoters of class II genes can be divided into four classes, and the combination of all
these cis-elements gives a promoter its characteristic strength. The first two classes,
TATA box and the initiator (Inr), are considered as the core promoter elements, and one
or both of them appears to be present in all protein-coding genes. The TATA box,
TATAAA, is located 25-30 base pairs upstream Of the transcription start site, and is
critical to determine the site of transcription initiation (Wasylyk et al., 1980; Grosschedl
and Birnsteil, 1980; reviewed by Breathth and Chambon, 1981). The initiator element
consists of 17 nucleotides encompassing the transcription start site, and is present in most

promoters, although its nucleotide sequence is not highly conserved (reviewed by Weis

and Reinberg, 1992). While TATA box and Inr can function cooperatively when present
together (Smale and Baltimore, 1989; Nakatani et al., 1990; Conaway et al., 1990), either
of them can potentially direct transcription initiation by itself (Myers et al., 1986; Smale
et al., 1990; Nakatani et al., 1990). The basal transcription machinery, consisting of
RNAP n and eras, functions through the TATA box and Initiator. The third class of cis-
promoter element provides binding sites for transcription factors that recognize specific
sequences such as CCAAT and the GC element. They are located within a few hundred
base pairs of the transcription start site. (McKnight and Kingbury, 1982; Myers et al.,
1986; Maniatis et al., 1987). The fourth class is enhancers, which may be located
thousands of base pairs away from the start site (reviewed by Khoury and Gruss, 1983).
The activators binding to enhancers can bind and stimulate the basal transcription
machinery through DNA looping and protein-protein contacts to regulate transcription
(reviewed by Ptashne, 1988; Ptashne and Gann 1990). Because their presence and
arrangement are gene-specific, both consensus sequence elements and enhancers are

considered as variable cis-elements.
Template Activation

Although biochemical studies of transcription by RNAP II have been carried out
predominantly with naked DNA terriplates, DNA in vivo is packaged into a nucleoprotein
complex known as chromatin. Packaging of naked DNA templates into chromatin causes
transcription to be repressed. The binding cf transcription activators and/or basal
transcription factors, such as TFIID, to the DNA template prior to the assembly of
chromatin can relieve this repression, suggesting that transcription activators function, at
least in part, to antagonize chromatin-mediated inhibition of transcription (Croston et al.,
1991; reviewed by Paranjape et a1. 1994). A nuclear multiprotein complex, known as the
SWIISNF complex, was observed to induce changes in chromatin structure, and facilitate

dramatically the binding of transcription activators or GTFs to the nucleosomal DNA
templates to allow transcription activation (Kwon et al., 1994; Imbalzano et al., 1994).

Promoter Recognition and Transcription Initiation

Accurate transcription by RNAP II is initiated from promoter sequences. RNAP II
and GTFs ('I'FIIA, TFIID, TFIIB, TFIIB, TFIIF, TFIIH, and TFIIJ) assemble on the core
promoter to form a pre-initiation complex, which is a highly ordered, stepwise process
(reviewed by Weinmann, 1992, Zawel and Reinberg, 1993; Conaway and Conaway,
1993; Buratowaki, 1994; Maldonado and Reinberg, 1995). Once assembled, DNA strands
are separated to expose the template for phosphodiester bond synthesis, and the pre-
initiation complex is converted into a transcriptionally active open complex. This step
requires hydrolysis of ATP at the 8-7 bond position (Bunick et al., 1982; Sawadogo and
Roeder, 1984). This energy requirement is unique to RNAP II transcription since RNA
polymerase I, III, and bacterial RNA polymerase do not require ATP hydrolysis for
transcription initiation. Although the mechanism involved in this requirement is still not
clear, evidence suggests that ATP might serve as a substrate for a helicase activity that
carries out the DNA unwinding reaction at the transcription start site (W. Wang, et al.,
1992). Alternatively, it was shown that ATP hydrolysis was required not during the
formation of the first few phosphodiester bonds in the RNA, but subsequently for
promoter escape (Goodrich and Tjian, 1994). Promoter escape, also called promoter
clearance, is the transition stage from abortive initiation to productive elongation. Newly
synthesized short RNA transcripts, 5 to 10 nucleotides in length, are not stably associated
with RNAP II and are released before productive elongation occurs (Luse and Jacob,
1987; Linn and Luse, 1991; Jacob et al., 1991; 1994). After transcription initiation, the
structure of the initiation complex is altered, so RNAP II can escape the promoter and
enter the elongation phase. It has been shown that TFIIE, TFIIH and ATP hydrolysis are

required for promoter escape in a reconstituted transcription system (Goodrich and Tjian,
1994). In cell extract systems the RAP74 subunit of TFIIF (Chang et al., 1993) and P-
TEFb (Marshall and Price, 1995) are additionally required for promoter escape. In the
reconstituted system, after promoter escape, TBP remained bound to the promoter,
whereas TFIIB, TFIIB, TFIIF, and TFIIH were released from the complex (Zawel et al.,
1995). In vivo, in the presence of a full complement of elongation factors, some of these

factors may remain associated with elongating RNAP II.

Elongation

Once RNAP II escapes the promoter, it commences highly processive elongation
until it encounters a pause, arrest or termination site. Elongation may be discontinuous
since RNAP 11 may stall without releasing the transcript. The barriers to elongation
include DNA sequences recognized by RNAP II, DNA-binding proteins and
nucleosomes. Protein factors that affect the DNA template structure and topology or
directly act on the ternary elongation complex (RNA-DNA-protein) can regulate
elongation. Elongation factors can be divided into at least two classes. The first class
includes SII/l‘FIIS, which helps RNAP II to read-through the template when it is arrested
in ternary complexes (reviewed by Kane, 1994; by Reines, 1994). THIS can stimulate the
cleavage of nascent RNA from the 3' end by RNAP II. This cleavage reaction causes
RNAP II to move backwards and releases a short RNA fragment of 7-17 nucleotides
(Rudd et al., 1994). Once the transcription is restarted from the newly exposed 3' end of
the transcript, RNAP II can move through the pause. The second class of elongation
factors includes TFIIF, which increases the overall rate of elongation and prortrotes more
rapid read-through of some blocks to elongation (Flores et al., 1989). It was proposed that
the stimulation of elongation by TFIIF is mediated by the phosphorylation of its RAP74

subunit (Kitajima et al., 1994). Another elongation factor, HIS, also stimulates the rate of

elongation and promotes the read-through (Bradsher et al., 1993; Bradsher et al., 1993b).
Transcription activators can also stimulate elongation by RNAP II in viva (Y ankulov et

al., 1994).

Termination

Transcription termination results when the RNA polymerase not only stops
transcription but also releases its transcript (reviewed by Kerppola and Kane, 1991). The
termination regions of DNA templates are downstream from the 3'-end processing
signals. RNAP II releases both its transcript and the DNA template when it crosses the
termination regions (Proudfoot, 1989). The position of termination regions is gene
specific and can span several hundred nucleotides (Wahle and Keller, 1992). The
sequences sufficient for polyadenylation are not sufficient for termination. Also required
is a region several hundred nucleotides downstream, where the termination occurs
('I‘antravahi et al., 1993).

The mechanism controlling RNAP II to terminate transcription is poorly
understood. RNAP II recognizes specific sites in the DNA template which serve as
termination signals. This recognition appears to be intrinsic to the polymerase as it occurs
in the absence of accessory factors. The intrinsic termination site usually consiSts of T-
rich region in the nontranscribed strand (Reines et al., 1987; Kerppola and Kane, 1988).
Sequences flanking the termination site have been suggested to influence intrinsic
termination efficiency. RNA secondary structures do not appear to control selection of
these sites, but DNA structure affects the efficiency of intrinsic termination, probably by
bending the template (Kerppola and Kane, 1990). RNA-binding proteins, which are
involved in accurate 3' end formation of RNA transcripts, may also influence the

termination reaction (Proudfoot, 1989);

Transcription Initiation by RNA Polymerase II

Transcription initiation by RNAP II is a complex process. Two working models of
pre-initiation complex formation on TATA-containing promoters have been proposed:

the ordered multistep assembly model and the holoenzyme assembly model (Fig. 1).

Multistep Assembly Process

In this model, the RNAP II and GTFs assemble on the TATA-containing
promoter in a highly ordered fashion through protein-DNA and protein-protein
interactions (reviewed by Zawel and Reinberg, 1993). In addition to RNAP II, at least
seven GTFs (TFIID, TFIIA, TFIIB, TFIIF, TFIIB, TFIIH, and TFIU) modulate basal
transcription. This process begins with the binding of TFIID to the TATA element. TFIID
is the only GTF that binds DNA specifically, and this function is intrinsic to one of its
subunits, the TATA binding protein (TBP) (Lewin, 1990). Other subunits of TFIID were
termed TAFs (TBP associated factors). TFIIA appears to have no essential role in basal
transcription (Cortes et a1. 1992), but can stabilize the binding of TFIID to the TATA box
(Buratowski et al., 1989; Maldonado et a1, 1990; Lee et al., 1992). The addition of TFIIB
to the DA complex (complex including promoter DNA, TFIIA and TFIID) results in a
DAB complex (Buratowsld et al., 1989; Maldonado et a1, 1990). TFIIB serves as a bridge
between TBP and RNAP II, and this interaction is critical for determination of the
transcription start site (Pinto et al., 1992; Berroteran et al., 1994; Li et al., 1994).
Although both TFIIB (Tschochner et al., 1992; Ha et al., 1993) and TBP (Usheva et al.,
1992) can bind directly to RNAP II, stable binding of RNAP II to the DAB complex
requires prior association of TFIIF with RNAP II (Flores et al., 1991; Buratowsk et al.,
1991). The RAP30 subunit of TFIIF reduces the affinity of RNAP II for non-specific
DNA (Killeen and Greenblatt, 1992), and by itself is capable of delivering

Figure 1. Preinitiation complex formation: the ordered multistep assembly model
and the holoenzyme assembly model

 

 

 

ll

RNAP II to the DAB complex (Flores et a1. 1991; Killeen et al., 1992). The RAP74
subunit of TFIIF likely stabilizes the complex (Flores et al., 1991; Tan et al., 1994;
Coulombe et al., 1994). After formation of the DABPolF complex, TFIIB, TFIIH and
TFIIJ sequentially join and form the complete DABPolFEHJ pre-initiation complex.

TFIIH contains several enzymatic activities, including an ATPase, a helicase and
a kinase specific for the carboxy-terminal domain (CTD) of the largest subunit of RNAP
II, and is essential to form a transcriptionally active complex (reviewed by Drapkin and
Reinberg, 1994). TFIIE is required for the stable association of TFIIH with the complex
(Flores et al., 1992), and stimulates the CPD kinase activity of TFIIH (Serizawa et al.,
1994; Ohkuma and Roeder, 1994). TFIIJ was proposed as the last GTFs to bind and
complete assembly of the pre-initiation complex (Zawel and Reinberg, 1993). The
requirement, identity and function of TFIIJ, however, are not clearly established, and
TFIIJ may be a component of native TFIID (Zawel and Reinberg, 1993).

Minimal Transcription Machinery

The multistep assembly model was derived from studies using a highly purified
reconstituted system in vitro. It appears that under certain conditions, some GTFs can be
dispensible for accurate transcription. Killeen et al. showed that TBP, TFIIB and the
RAP30 subunit of TFIIF are sufficient for promoter recognition by RNAP II (Killeen et
al., 1992). Tyree et a1. further demonstrated that recombinant TBP, TFIIB, and RAP30,
along with purified, native RNAP II, are able to accurately transcribe a subset of
promoters using supercoiled templates (Tyree et al., 1993). Therefore TBP, TFIIB,
RAP30 and RNAP II constitute a central, catalytic core of the transcription machinery.
Negative supercoiling of the template removes the requirement for DNA strand
separation by TFIIH and TFIIB (Goodrich and Tjian, 1994). The requirement for certain
GTFs is also promoter dependent. When a supercoiled template was used, transcription of

12

the IgH promoter required only TBP, TFIIB, and RNAP II (Parvin and Sharp, 1993).
Only TFIIB, RNAP II, and the W1 transcription factor were required for initiation from
supercoiled adeno-associated virus P5 promoter (U sheva and Shenk, 1994). Thus, it
appears that only TFIIB, RNAP II, and a factor (TBP or YYl) that binds DNA at the

TATA box or Initiator are absolutely required for accurate, basal level initiation in vitro.
Holoenzyme Assembly Model

Although it has been generally accepted that the assembly of the pre-initiation
complex is a highly ordered multistep process, more recent evidence suggests that there is
another pathway for assembly, in which RNAP II bound with a much more complex set
of GTFs and accessory proteins binds to the promoters. This more complex form of
RNAP II is refered to as the RNAP II "holoenzyme”. TFIIB, TFIIF, TFIIB, and TFIIH
assemble with RNAP II to form a transcriptionally active multifunctional complex
capable of binding directly to TBP at the promoter in a single step (Serizawa et al.,
1994b). A yeast RNAP 11 holoenzyme containing TFIIB, TFIIF, TFIIH, and many other
unidentified polypeptides has been isolated from cell extracts by Young and co-workers
(Koleske and Young, 1994; reviewed by Koleske and Young, 1995). This holoenzyme is
capable of accurate transcription initiation when supplemented with TBP and TFIIB.
Using another approach, Kornberg and co-workers have isolated another yeast RNAP II
holoenzyme, containing RNAP II, SRB (suppressor of RNA polymerase B) proteins,
Gall 1, sugl, TFIIF and other unidentified polypeptides (Kim et al., 1994). This
holoenzyme stimulates basal transcription and also affects the CFD kinase activity of
TFIIH. This preparation can be further separated into two fractions: one consists of 12
core subunits of RNAP II and can support basal transcription, the other one, which
stimulates both basal and activated transcription and was termed the "mediator", contains

SRBs, Gall 1, Sugl, TFIIF and other unidentified polypeptides (Kim et al., 1994).

13

Taken together, these results suggest that the preinitiation complex may be able to
assemble by different pathways on different promoters. Several forms of RNAP 11
holoenzyme may exist in cells for transcription of different genes. Since the multistep
assembly pathway was established based on the minimal requirement for assembly in

vitro, the holoenzyme pathway is expected to more closely resemble the physiological
function of RNAP II.

Transcription Activators

Transcription activators are a group of regulatory proteins that bind to the specific
, sequences located upstream or downstream of the core promoter region and increase the
rate-of transcription initiation. Transcription activators contain at least two domains: a
DNA-binding domain that recognizes specific sequences, and a separate and
independently acting domain required for activation (Ptashne, 1988). Based on the
properties of their activation domains, activators have been loosely classified into several
groups, such as acidic (including the yeast activator Gal4 and herpes simplex virion
protein VP16), glutamine-rich (including Spl) and proline-rich (including CTF)
(reviewed by Mitchell and Tjian, 1989; by Tjian and Maniatis, 1994). Of these classes,
acidic activators are unique in that they can function universally in all eukaryotes.

It is widely believed that activators function to facilitate some rate-limiting step in
complex assembly or initiation by interacting with basal transcription factors and
mediators. Some of the GTFs have been shown to be the target of upstream activators.
VP16 can bind to the TBP component of TFIID (Stringer et al., 1990). Mutations that
affect the strength of the activator also affect its ability to bind TFIID (Ingles et al.,
1991). TFIIB also binds to the activation domain of VP16. A critical mutation in the
activation domain of VP16 that dramatically reduces the activation potential eliminates

binding to TFIIB (Lin and Green, 1991). Mutations in TFIIB that disrupt the interaction

14

with VP16 also cause defects in activated transcription but function normally in basal
transcription (Roberts et al., 1993).

TFIIB has two functional domains: an N-terminal domain that interacts with
TFIIF and RNAP II, and a C-terminal domain that interacts with TBP (Ha et al., 1993). It
was proposed that these domains are engaged in an intramolecular interaction, and the
binding of VP16 to the C-terminal domain of TFIIB disrupts this interaction and exposes
binding sites for TFIIF and RNAP II. In this way interaction with an activator promotes
pre-initiation complex formation (Roberts and Green, 1994) .

The 62kDa subunit of TFIIH is also the target of several activators, including
VP16 and the tumor suppressor p53 (Xiao et al., 1994). Since TFIIH is involved in
transcription, nucleotide excision repair, and perhaps cell cycle progression (Drapkin and
Reinberg, 1994), the interactions between TFIIH and activators could be important not
only in uanscription, but also in other cellular process.

Coactivators

Although transcription activators can directly interact with GTFs, addition of
transcription activators to highly pmified reconstituted transcription systems does not
substantially stimulate transcription. Activation, therefore, may require additional
components to fully transduce the activation signal from the activator to the basal
machinery. Indeed, several of these factors, termed coactivators, mediator or adaptors,
have been identified. The coactivators can either enhance (PCI, PC2, PC3, PC4, ACF) or
repress (NCl, NC2/Drl, Dr2/PC3/T0poisomerase I (Topo I), MOTI/ADI, NOT1-4,
TUP) transcription, and their functions appear to be general, in the sense that these factors
do not appear to bind specifically to promoter DNA (reviewed by Zawel and Reinberg,
1995).

15

Of these coactivators, the best known ones are Dr2IPC3/I‘opo I and PC4. Topo I
interacts with TBP and interferes with the ability of TFIID to bind to the TATA box,
repressing basal transcription. However, Topo I can also enhance the response of the
basal transcription complex to activators, and since repression is overcome by activators,
the net fold-activation is greater in the presence of Topo I (Merino et al., 1993;
Kretzschmar et a1, 1993). Indeed, Topo I was first isolated by tracking an activity that
enhanced the response to activators (Merion et al., 1993), and was also considered as a
positive cofactor, PC3 (Kretzschmar etal., 1993). Among positive cofactors PC1-PC4,
PC4 is the best characterized. PC4 binds to single stranded DNA and its transcriptional
activity is negatively regulated by phosphorylation by casein kinase II (Kretzschmar et
al., 1994; Ge et al, 1994). PC4 does not affect basal transcription, but rather enhances the
response to multiple activator types. Consistent with this function, PC4 was found to
interact with both the VP16 activation domain and DNA-TBP-TFIIA complex (Ge and
Roeder, 1994; Ge et al., 1994).

RNA Polymerase II

RNAP II has been purified from several eukaryotic species, and generally has 10-
12 subunits with sequences and structures that are similar between species. HeLa cell
RNAP 11 contains 10 subunits ranging in size from 240 to 10 kDa. The yeast RNAP II,
which appears to have 10 subunits with apparent molecular sizes from 220 to 10 kDa in
SDS-PAGE, is actually composed of 12 subunits, RPBl-RPB12, and the genes encoding
all these 12 subunits have been cloned and shown to be essential for wild type growth
(reviewed by Young, 1991; by Woychik and Young, 1994). The three largest subunits,
RPBl, RPB2, and RPB3, have counterparts in the bacterial RNA polymerase. Amino
acid sequence comparison shouted that RPBl and RPB2 are clearly related to the two
largest subunits of E. cali RNA polymerase, 13' and B, respectively (Allison et al., 1985;

l6

Sweetser et al., 1987). RPBl and RPB2 are also functional homologs of their bacterial
counterparts. RPBl, like 5', is involved in DNA-binding whereas both RPB2 and B bind
nucleotide substrates (reviewed by Young, 1991). The third largest subunit, RPB3, is
related to the a subunit of E. cali RNA polymerase (Kolodziej and Young, 1991; Young,
1991). The two largest subunits of RNAP II are also related in size and sequence to the
two largest subunits of RNA polymerases I and III, whereas RPBB and RPBll are
homologous to the AC40 and AC19 subunits of RNA polymerases I and III (Mann et al.,
1987; Woychik and Young, 1994). In addition, all three nuclear RNA polymerases share
five identical subunits, the RPBS, RPB6, RPB8, RPB 10, and RPBlZ (Woychik et a1,
1990, Carles et al., 1991; Treich et al., 1992).

The three eukaryotic nuclear and RNA polymerases have significant structural
and functional relationships with bacterial RNA polymerase. The largest subunit of
RNAP 11, however, contains a unique carboxy terminal domain (CID) that is not present
in its prokaryotic counterpart or in RNA polymerases I or III (reviewed by Dahmus,
1994). The CTD consists of multiple repeats of the consensus sequence YSPTSPS. This
heptapeptide sequence is repeated 26-27 times in yeast, 42-44 times in Drasaphila, and
52 times in mouse and human. The CI'D can be highly phosphorylated at serine and
threonine sites in the heptapeptide repeats. The highly phosphorylated form of RNAP II,
which is 240 kDa, is referred to as the 110 form, and the unphosphorylated or
dephosphorylated form of 215 kDa as the HA form (Cadena and Dahmus, 1987; Kim and
Dahmus, 1989). The third form, 11B, which is 180 kDa and lacks most or all of LTD, is
only observed in vitro and is considered to be a proteolytic product of purification (Kim
and Dahmus, 1988; Kolodziej et al., 1990).

Genetic analysis indicates that the CTD has an essential role in viva. Deletion
mutations that remove most or all of the CPD are lethal in yeast (Nonet et al., 1987;
Allison et al., 1988), Drasaphiia (Zehring et al., 1988) and mouse (Bartolomei et al.,
1988). However, the dependency on the CPD appears to be promoter-specific. In yeast

l7

truncations of the CTD from 27 to 11 repeats do not significantly reduce transcription
from the [-1154 gene whereas the inducible transcription of the [NO] and GALIO genes is
eliminated (Scafe et a1, 1990). In vitro transcription studies also showed that CTD is
unnecessary for Spl- or Ad-2 MLTF-mediated activation (Buratowski and Sharp, 1990;
Zehring and Greenleaf, 1990). The simplest interpretation of these results is that RNAP II
is recruited to the promoter by a variety of interactions and only some of these involve
the CTD. Several models for CTD function have been proposed, including involvement
in transcription initiation, enzyme localization, DNA-binding, removal of chromatin
proteins from DNA, and general regulation of enzyme activity (Young, 1991). A longer
CTD seems to complement Gal4 deletion mutations (Allison and Ingles, 1989), and the
CID can directly interact with the TBP of TFIID (U sheva et al., 1992; Conaway et al.,
1992). Thus it seems likely that CTD functions to facilitate interactions between upstream
transcription activators and components of the basal transcription machinery, either
directly or through additional factors.

The interaction of RNAP II with the preinitiation complex is dramatically
influenced by the state of phosphorylation of the CTD. The nonphosphorylated 11A form
stably associates with the preinitiation complex, whereas the phosphorylated [10 form
associates with the actively elongating complex. The conversion of RNAP IIA to RNAP
no occurs prior to the formation of the first phosphodiester bond (Cadena and Dahmus,
1987; Payne et al., 1989; Laybourn and Dahmus, 1989; Layboum-and Dahmus, 1990; Lu
et al., 1991). The postulated interaction between the acidic activation domains of a
transcription activator and the CTD could be disrupted by phosphorylation of the CTD,
providing a switch from initiation to elongation (Suzuki, 1990; Peterson et al., 1991). A
model was presented that phosphorylation of CTD modulates the transition from
initiation to elongation (Laybourn and Dahmus, 1990; Buratowski and Sharp, 1990). The
HA form of RNAP 11 would associate with assembly preinitiation complexs, then CTD

kinases would trigger the elongation phase by phosphorylating the CTD.

18

The CTD phosphatase acts to reset the cycle. CI'D kinase is an intrinsic activity of
TFIIH (Feaver et al., 1991; Serizawa et al., 1992; Lu et al., 1992). A Cl‘D phosphatase
has also been identified (Chambers and Dahmus, 1994). This phosphatase interacts with a
region of RNAP II distinct from CI'D, and will not dephosphorylate the CTD unless this
domain is covalently linked to RNAP II. The RAP74 subunit of TFIIF stimulates CTD

phosphatase activity, and this stimulation can be suppressed by TFIIB (Chambers et al.,
1995).

General Transcription Factors

TFIID (TBP and TAFs)

TFIID was first identified as an activity in nuclear extracts that can specifically
bind a TATA-containing promoter (Parker and Topol, 1984). Further characterization
showed that native TFIID is a multiprotein complex composed of the TATA-box-binding
protein (TBP) and a number of tightly bound proteins called TBP-associated factors
(TAFs) (reviewed by Hernandez, 1993; Goodrich and Tjian, 1994b; Moncollin et al.,
1994). The data from biochemical and genetic studies suggest that TBP is involved in
transcription by all three RNA polymerases (reviewed by Hernandez, 1993; Rigby,
1993). TBP is a subunit of the RNA polymerase I transcription factor SL1 (Comai et al.,
1992) and a subunit of the RNA polymerase III transcription factor TFIIIB (Huet and
Sentenac, 1992; Kassavetis et al., 1992; Lobo et al., 1992; Simmen et al., 1992; Taggart
et al., 1992; White and Jackson, 1992). In yeast, mutations in TBP affect transcription by
all three RNA polymerases (Cormack and Struhl, 1992; Schultz et al., 1992). Thus, TBP
is a universal eukaryotic transcription factor involved in the formation of the initiation
_ complexes on TATA-containing and TATA-less RNA polymerase I, II, and III
promoters.

19

cDNAs encoding TBP have been cloned from a number of species, including
yeast (Cavallini et al., 1989; Hahn et al., 1989; Horikoshi et al., 1989; Schmidt et al.,
1989), Drasaphila (Hoey et al., 1990; Muhich et al., 1990) and human (Hoffman et al.,
1990; Kao et al., 1990; M.G. Peterson et al., 1990). TBP is a small protein of 27 kDa in
yeast, and 38 kDa in Drasaphila and human. Amino acid sequence comparison of TBP
from different species shows that the N-terminal region is highly divergent and the C-
terminal region is highly conserved. The C-terminal region invariably contains two
copies of a long imperfect repeat of 61-62 amino acids and short basic repeats
(Hernandez, 1993). Although yeast and human TBP are highly conserved at the C-
terminal domain and can substitute for each other in basal transcription in vitro, human
TBP is unable to functionally replace yeast TBP in viva (Cormack et al., 1991; Gill and
Tjian, 1991). The analysis indicates that the species specificity is due to many minor
differences within the conserved C-terminal domain, instead of differences in the highly
divergent N-terminal domain. Mutagenesis demonstrates that the conserved C-terminal
domain is sufficient for binding to the TATA box and basal transcription activities
(Horikoshi et al., 1990; Hoey et al., 1990; M.G. Peterson et al., 1990), whereas the
function of the divergent N-terminal region is unclear, although there are some
indications that it may mediate some interactions with coactivators (Pugh and Tjian,
1990; Zhou et al., 1991).

TBP interaction with its TATA box recognition sequence has been analyzed in
servers] ways. Kinetic analysis of yeast TBP-TATA box complex formation suggests a
two-step pathway. The first step includes the binding of TBP to the TATA box, followed
by the formation of a stable TBP-TATA complex likely dependent on conformational
changes in the TBP, promoter, or both (Hoopes et al., 1992). TBP, as a monomer
(Horikoshi et al., 1990), contacts DNA through interactions with the minor groove of the
TATA element (Starr and Hawley, 1991; Lee et al., 1991), and induces a 100° bend
around the TATA element (Horikoshi et al., 1992; Y. Kim et al., 1993; IL. Kim et al.,

20

1993). X-ray crystallography studies predict that TBP resemble a saddle in which the C-
terminal direct repeats straddled the DNA (Nikolov et al., 1992; Y. Kim et al., 1993; IL.
Kim et al., 1993). The orientation of TBP binding to DNA exposes the entire upper
surface, of TBP, and provides a target for many protein-protein interactions. Indeed, a
diverse set of proteins, including GTFs, u'anscription activators and coactivators, have
been reported to bind TBP specifically.

TBP is tightly associated with TAFs, which can only be dissociated under
denaturing conditions. At least 7 TAFs have been identified from human (Pugh and
Tjian, 1991; Zhou et al., 1993), 8 from Drasaphila (Dynlacht et al., 1991). 9 yeast TAFs,
which are more weakly associated with TBP, have also been identified (Reese et al.,
1994). Monoclonal antibodies against the CTD of RNAP II block transcription initiation
by native TFIID, but not by TBP, suggesting that TAFs may function to establish
interactions between RNAP II and TFIID (Conaway et al., 1992). One or more TAFs may
interact directly with TFIIH to stabilize its interaction with the preinitiation complex
(Conaway et al., 1992b). However, the major function of TAFs appear to serve as
coactivators that provide a link between the activation domains of enhancer-binding
activators and the basal transcription machinery to direct gene-specific transcriptional
activation (Chiang et al., 1993; Chen et al., 1994; Jacq et al., 1994). Specific interactions
between TAFs and activators have been demonstrated, e.g., dTAF40 interacts with VP16
(Goodrich et al., 1993), dTAFl 10 with Spl (Hoey et al., 1993), and dTAF150 with NW-
1 (Chen et al., 1994). Different activators require different subsets of TAFs, e. g., a
complex containing only TBP, TAF150 and TAF250 is sufficient to mediate a response
to NTP-1 (Chen et al., 1994). Recent studies suggest that TAFs may play an important
role not only as coactivators, but also as core promoter recognition factors. TAF150 was
found to have sequence-specific DNA-binding activity at select core promoters (Verrijzer
et al., 1994). Further investigation found that TAF250 and TAF150 can confer promoter

selectivity by interacting specifically with downstream elements that include the initiator,

21

increasing the stability of TBP binding to TATA box, and mediating a higher level of

transcription (Verrijzer et al., 1995).
TFIIA

TFIIA was first identified as a heterotrimer composed of three subunits of 37 kDa
(a), 19 kDa (B), and 13 kDa (y) from mammalian cells (Cortes et al., 1992), and a
heterodimer composed of two subunits of 32 kDa and 13 kDa from yeast, which are
encoded by the TOAI and TOAZ genes, respectively (Ranish et al., 1992). The isolation
of cDNA encoding human and Drasaphiia TFIIA revealed that the a and B subunits are
actually encoded by a single cDNA, and therefore are probably generated by protein
processing (Ma et al., 1993; DeJong and Roeder, 1993; Yokomori et al., 1993). Amino
acid sequence analysis showed that the Drasaphila and human (1 and B subunits share
sequence similarity with the amino- and carboxy terminal regions of the large subunit of
yeast TFIIA, and the 1 subunit is homologous to the small subunit of yeast TFIIA (Ma et
al., 1993; DeJong and Roeder, 1993; Yokomori et al., 1993; Sun et al., 1994; Yokomori
et al., 1994; Ozer et al., 1994; Bernstein et al., 1994). Human and Yeast TFIIAs are
functionally exchangable in basal transcription-(Rarrish et al., 1992).

TFIIA functions at an early stage of pre-initiation complex formation (Reinberg et
al., 1987; Flores etal., 1992), by stimulating the binding of TFIID to DNA through direct
interaction with TBP (Buratowski etal., 1989; Maldonado et al., 1990; Lee et al., 1992;
Yamamoto et al., 1992; Buratowski and Zhou, 1992). TFIIA stimulates basal
transcription when native TFIID is used, but has no effect when TBP is used. It was
proposed that TFIIA functions as an anti-repressor, through interaction with TBP, to
remove the negative components present in the crude TFIID fraction (Cortes et al., 1992).
Further investigation demonstrated that TFIIA indeed counteracts the activity of Dr2, a
negative regulator of basal transcription associated with TFIID (Merino et al., 1993).

22

However, transcription reactions useing highly purified TFIID, which does not contain
repressors, are still significantly stimulated by recombinant TFIIA, suggesting that TFIIA
directly stimulates transcription, perhaps through a TAF (Sun et al., 1994; Yokomori et
al., 1994; Ozer et al., 1994; Bernstein et al., 1994). Indeed, TFIIA interacts directly with
Drosophila TAF110 (Yokomori et al., 1993). TFIIA can also stimulate aetivated
transcription by several activators, including Spl, NTF-l, and VP16 (Yokomori et al.,
1994). TFIIA binds directly to Zta and supports transcription activation by this activator
(Ozer et al., 1994). Positive coacdvator PC4 interacts with VP16, as well as TFIIA within
the DNA-TBP-TFIIA complex (Ge and Roeder, 1994; Ge et al., 1994).

TFIIB is a single polypeptide of 33 kDa in human (Ha et al., 1991), 34.5 kDa in
Drosophila (Wampler and Kadonaga, 1992), and 35 kDa in yeast (Pinto et al., 1992). The
structure of TFIIB can be divided into two domains: a compact. protease-resistant C-
terminal core, and a protease-susceptible N-terminal region (Barberis et al., 1993; Malik
et a1, 1993). Mutagenesis showed that the entire C-terminal core, which contains two
imperfect direct repeats and a region with the potential to form an amphipathic helix
flanked by the repeats (Ha et a1, 1991; Pinto et al., 1992), is required and sufficient to
interact with the promoter-bound TBP, and mutations in this region abolish the ability of
TFIIB to support basal transcription (Buratowski and Zhou, 1993; Ha et a1, 1993;
Hisatake et a1, 1993; Yamashita et al., 1993). The N-terminal region, which contains a
putative zinc finger motif (Ha et a1, 1991; Pinto et al., 1992), is important to recruit
RNAP II to the promoter (Barberis et al., 1993; Buratowski and Zhou; Ha et al., 1993;
Hisatake et al., 1993), and to bind the RAP30 subunit of TFIIF (Ha et al., 1993).
Sequences comparison of TFIIB from different species revealed a highly conserved

23

region immediately distal to the zinc finger (Na and Hampsey, 1993), which is required
for accurate start-site selection in viva (Pinto et al., 1994).

TFIIB plays a critical role in basal transcription initiation. It is a component of
minimal transcription machinery (Tyree et al., 1993; Parvin and Sharp, 1993; Usheva and
Shenk, 1994). In the absence of other u'anscription factors, TFIIB is capable of binding
stably to the TBP-promoter complex to form the DB complex (Buratowski et al., 1989).
TFIIB also directly interacts with RNAP II (Wampler and Kadonaga, 1992; Tschochner
et al., 1992; Ha etal., 1993). Thus, TFIIB serves as a bridge between RNAP II and TBP.
In initiation complex assembly, the association of TFIIB is a rate-limiting step (Lin and
Green, 1991). The crystal structure of TATAbox/l'BP/I‘FIIB was recently solved, and the
results strongly suggest that the N-terminal region of TFIIB is presented as a scaffold for
subsequent assembly of RNAP II/TFIIF into the pro-initiation complex (N ikolov et al.,
1995). Nuclear magnetic resonance structure of TFIIB also supports this model (Bagby et
al., 1995). Genetic studies suggest that the interaction between RNAP II and TFIIB is
critical in determining the transcription start site. Mutations in the yeast gene encoding
TFIIB (SUA7) shifted the transcription start site (Pinto et al., 1992). Mutations in the
largest subunit of RNAP II similarly affected initiation, suggesting that there is a
functional interplay between TFIIB and the largest subunit of RNAP II in determining the
transcription start site (Berroteran ct al., 1994). Biochemical data are consistent with this
suggestion. In a transcription system reconstituted with Saccharamyces cerevisiae GTFs,
only pair-wise exchange of RNAP II and TFIIB from S. cerevisiae with counterparts from
Schizasaccharamyces pambe shifted transcription initiation to an initiation pattern
characteristic of S. pambe (Li et al., 1994). Thus, RNAP II and TFIIB are the sole
determinants of the transcription start site.

TFIIB is also involved in activated transcription. The acidic activators GAL4-
VP16 and GALA-AH, and the proline-rich activator GAL4-CTF can function to recruit
TFIIB and/or stabilize its association with TFIID (Lin and Green, 1991; T.K. Kim and

24

Roeder, 1994). A direct interaction between TFIIB and VP16 is required for
transcriptional activation, and mutations in TFIIB that disrupt this interaction reduced
activated, but not basal, transcription (Roberts et al., 1993). The N-terminus of TFIIB was
proposed to bind to the C-terminus via an intramolecular interaction, and the binding of
VP16 to the C-terminal region disrupts this interaction, changing the conformation of
TFIIB in favor of formation of the preinitiation complex (Roberts and Green, 1994).
Recently, a glutamine-rich activator GAL4-ftzQ was demonstrated to directly interact
with the N-terminus of TFIIB to mediate u'anscriptional activation, suggesting that
different activators can function by contacting distinct regions of TFIIB (Colgan et al.,
1995).

TFIIE is a heterotetramer composed of two 56 kDa (a) and two 34 kDa (B)
subunits (Ohkuma et al., 1990; Inostroza et al., 1991; M.G. Peterson et al., 1991). cDNAs
encoding both subunits of TFIIB haves been isolated (M.G. Peterson et al., 1991;
Ohkuma et al., 1991; Sumimoto et al., 1991). The N-terminal region of TFIIB-a shows
similarity to the a subunit of bacterial RNA polymerase and several interesting structural
motifs: a leucine repeat, a zinc finger, and a helix-turn-helix (Ohkuma et al., 1991).
Recent mutational analysis suggests that the N-terminal half of TFIIB-a is essential for
transcription and phosphorylation. The regions containing the leucine repeat and helix-
turn-helix are important for interaction with TFIIB-B, and the zinc finger domain may be
important for interaction with RNAP n (Ohkuma et al., 1995). The acidic C-terminal half
is a direct binding site for TFIIH and mutation of this site also reduces basal transcription
dramatically (Ohkuma et al., 1995). TFIIB-B contains regions similar to bacterial 0
factors and the RAP30 subunit of TFIIF, and a portion of the basic region-helix-loop-
helix motif found in several enhancer-binding proteins (Sumimoto et al., 1991).

25

TFIIB binds stably to RNAP II in solution and was purified as one of the RAPs
from an RNAP II affinity column (Flores et al., 1989; Buratowski et al., 1991). Further
study showed that it is TFIIB-a that binds to RNAP IIA (Maxon et al., 1994). The
association of TFIIB with the pie-initiation complex requires the DABPolF complex, and
form the DABPolFE complex, which is necessary for subsequent recruitment of TFIIH
(Flores et al., 1992). Many lines of evidence indicated that TFIIB functionally interacts
with TFIIH. TFIIB stimulates CTD phosphorylation by TFIIH (Lu et al., 1992; Ohkuma
and Roeder, 1994). TFIIB-a also mediates the helicase activity of TFIIH either negatively
(Drapkin et al., 1994) or positively (Serizawa et al., 1994). TFIIB has also been
implicated in promoter clearance in conjunction with TFIIH (Goodrich and Tjian, 1994).
TFIIB from Saccharamyces cerevisiae could not replace the Schizasaccharamyces pambe
TFIIB in an S. pambe system unless the S. cerevisiae TFIIE and TFIIH were swapped
together (Li et al., 1994). Indeed. TFIIB has been demonstrated to physically interact with
TFIIH as well as TFIIF (Maxon et al., 1994).

TFIIH is the most complex of the GTFs and the only GTF that has been shown to
contain enzymatic activities. The human TFIIH consists of 8 subunits of 34, 38, 41, 44,
50, 62, 80, and 89 kDa (Schaeffer et al., 1994). Its yeast homolog, factor b, contains 5
subunits (Feaver et al., 1993). TFIIH has several enzymatic activities, including an
ATPase, a helicase, and a kinase specific for the CFO of the largest subunit of RNAP II
(reviewed by Drapkin and Reinberg, 1994).

Most of subunits of TFIIH have been cloned from human, rat and yeast cells
(reviewed by Drapkin and Reinberg, 1994; by Maldonado and Reinberg, 1995). The
largest subunit of TFIIH, p89 in human, is identical to the nucleotide excision repair
protein XPB/ERCC3 , which contains ATP-dependent DNA unwinding helicase activity

26

(Schaeffer et al., 1993). Later, other subunits of TFIIH were identified as the products of
genes XPDIERCCZ and SSLI , which are also involved in nucleotide excision repair
(Feaver et al., 1993; Drapkin et al., 1994; Humbert et al., 1994). Like ERCC3, ERCC2
also contains ATP-dependent helicase activity. Purified TFIIH was found to be able to
restore nucleotide excision repair activity in a cell-free extract defective in nucleotide
excision repair (Drapkin et al., 1994; Wang et al., 1994; VanVuuren et al., 1994).
Recently, a reconstituted system for nucleotide excision repair with purified human
components was developed for the first time and it was completely dependent on TFIIH
(Mu et al., 1995). These results clearly demonstrate that TFIIH plays a role in nucleotide
excision repair. It was proposed that there are two forms of TFIIH, a holo-TFIIH
containing CTD-kinase activity and involved in transcription, and a repariasome lacking
CTD-kinase activity and active in nucleotide excision repair (Svejstrup et al., 1995).
Genetic and biochemical analyses of Rad3 (ERCC2) and Rad25 (ERCC3) in yeast
have defined the role of the ATPase and helicase domains of these proteins. The ERCC2
and ERCC3 helicase activities are required for nucleotide excision repair. However,
although both proteins are absolutely required for transcription, the ERCC2 helicase is
dispensable for this process, whereas the ERCC3 helicase is essential (Sung et al., 1988;
Feaver et al., 1993; Guzder et al., 1994). The helicase activity of TFIIH is involved in
promoter escape (Goodrich and Tjian, 1994). It was recently reported that two
polypeptides of TFIIH are the MOIS/Cdk7 kinase and cyclin H subunits of the Cdk
(cyclin-dependent kinase)-activating kinase Cak, which phosphorylates cdc2, Cdk2 and
Cdk4, and which is necessary for cell-cycle progression (Serizawa et al., 1995;
Shiekhattar et al., 1995). Thus, Cak is the CTD kinase intrinsic to TFIIH. In addition to
phosphorylating the CTD, TFIIH also specifically phosphorylates TBP, TFIIB-ct and the
RAP74 subunit of TFIIF (Ohkuma and Roeder, 1994). The presence of Cak in TFIIH
suggests that there could be a link between transcription and the cell cycle machinery.

The functional role of the CTD kinase of TFIIH, however, is not yet completely clear.

27

Since TFIIH plays multiple roles in transcription, nucleotide excision repair, and perhaps
cell cycle progression, it is likely that TFIIH is a target of regulators. Indeed, the p63
subunit of TFIIH interacts with the activation domains of VP16, and the tumor suppressor
p53 (Xiao et al., 1994).

TFIIF (RAP30/74)

RAP30/74 was first identified and purified from HeLa cell extract by affinity
chromatography on a column containing immobilized RNA polymerase 11, among a
group of proteins termed RAPs (RNA polymerase II-Associated Doteins), capable of
binding to immobilized RNAP II (Burton et al., 1986). Later it was purified to
homogeneity from rat liver as initiation factor B7 (Conaway and Conaway, 1989), from
Drosophila as factor 5 (Price et al., 1989), from human cells as TFIIF (Flores et al., 1988;
1990) and FC (Kitajima et al., 1990), and from yeast as factor g (Henry et al., 1992).
Human RAP30/74 is composed of two subunits with apparent relative molecular masses
of 30,000 and 74,000 (RAP30 and RAP74 subunits, respectively} (Burton et al., 1988).
Its activity elutes from a gel filtration column with an apparent molecular weight of 220
kDa, suggesting that it exists in solution as an (1sz structure (Flores et al., 1988).
Different from other TFIIF homologs, purified yeast factor g comprises three
polypeptides, with apparent masses of 105, 54, and 30 kDa (Henry et al., 1992).

The human cDNAs encoding RAP30 (Sopta et al., 1989) and RAP74 (kaelstein
et al., 1992; Aso et al., 1992) have been cloned. RAP30 and RAP74 produced in E. cali
can replace natural human RAP30fl4 to direct accurate transcription in vitro (F‘mkelstein
et al., 1992). The sequence of RAP30 cDNA encodes a protein of 249 amino acids with a
predicted relative molecular mass of 28,378 and is very basic (pI=10.4), with a region of
dense positive charge at amino-acid residues 168-182. It was suggested that the central
portion of RAP30 (residues 93-165) is weakly similar to two noncontiguous regions of E.

28

coli 070, the regions 2 and 1b. These two regions are the regions conserved among
bacterial and bacteriophage 0 factors. They are postulated to contain the site of
interaction of 670 with the core component of bacterial RNA polymerase (Sopta et al.,
1989). Another research group also suggested that the carboxy terminus of the RAP30
has weak sequence similarity with 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
subtiiis 0K) (Garrett et al., 1992). Region 4 is believed to direct recognition and binding
of RNA polymerase at the -35 element of bacterial promoters. These similaries, however,
are very weak.

The cDNA of RAP74 encodes a protein of 517 anrino acids with relative
molecular mass of 58, 254. RAP74 is a highly charged protein. The amino acid sequence
suggests that RAP74 has a globular N-terminal domain (residues 1-179), a charged
central domain (residues 180-356), and a globular C-terminal domain (residues 357-517)
(Finkelstein et al., 1992). Encompassing the end of the central domain and the beginning
of the C-terminal domain of RAP74 is a sequence, DSSEES, which is repeated three
times. The function of this sequence is unknown. It might serve as a target for' a serine -
kinase (Karlin, 1993).

Homologues of human RAP30 have been cloned from rat (Kobayashi et al., 1992;
Garrett et al., 1992), Xenapus (Gong et al., 1992), Drosophila (Gong et al., 1995; Frank et
al., 1995), and yeast ('1‘ng) (Henry et al., 1994). Sequence comparison reveals that
compared with human RAP30, rat RAP30 shows 97.6% identity, Xenapus RAP30 83%
identity and 91% similarity, Drosophila RAP30 50% identity and 63% identity, yeast
ng2 30.6% identity and 50.8% similarity. The homologues of human RAP74 have also
been cloned from Xenapus (Gong et al., 1992b), from Drosophila (factor 5) (Kephart et
al., 1993), and from yeast (SSU71/T‘fg1) (Henry et al., 1994; Sun and Hampsey, 1995).
Compared with human RAP74, Xenopus RAP74 shows 76% identity and 86% similarity,
Drosophila RAP74 43% identity and 65% similarity, and yeast Ssu7 l/ngl 26.6%

29

identity and 49.9% similarity. The conserved amino acids are found particularly in
regions near the amino and carboxyl termini of RAP30 or RAP74.

Yeast TFIIF is composed of three subunits, encoded by genes TFGI , TFGZ, and
TFG3, respectively. While ng1 and ng2 are the homologs of mammalian RAP74 and
RAP30, ng3, which is less tightly associated and at most stimulatory to transcription and
dispensable for cell viability, is one of TAFs, TAF30, and its gene is identical to a known
gene, ANCI, which is implicated in cytoskeletal function (Welch and Drubin, 1994;
Henry et al., 1994). ng3 has no known counterpart in mammalian TFIIF, but has
sequence similarity to human proteins ENL and AF-9 (Henry et al., 1994).

Antibodies against RAP30 co-immunoprecipitate RAP74, indicating that RAP30
and RAP74 are normally associated in a complex (Burton et al., 1988). The N-terminus
of RAP30 is essential and sufficient for RAP74 binding (Y onaha et al., 1992; Frank et al.,
1995; Tan et al., 1995; Chapter III), and the N-terminus of RAP74 interacts with RAP30
(Yonaha et al., 1992; Wang and Burton, 1995). Both subunits of RAP30fl4 are required
for accurate transcription in a HeLa cell extract, from which RAP30/74 has been depleted
by antibody against RAP30 (Burton et al., 1986, 1989). This result was confirmed with
systems reconstituted from purified components (Flores etal., 1989). Both RAP30 and
RAP74 are physical components of the pro-initiation complex (Flores et al., 1991). A
variety of evidence suggests that RAP30/7 4 promotes transcription initiation by a
mechanism similar to that of bacterial 0 factors. First, RAP30fl4 promotes binding of
RNAP II to the DAB complex (Flores etal., 1991). Second, RAP30/74 binds stably to
RNAP in solution (Reinberg and Roeder, 1987). Third, RAP30/74 prevents non-specific
binding of RNAP II to free DNA (Conaway and Conaway, 1990; Killeen and Greenblatt,
1992).

It was proposed that RAP30fl4 binds RNAP 11 through RAP30. Indeed, RAP30
appears not only structurally, but also functionally, homologous to bacterial 0 factors. It
was found that mammalian RNAP a specifically protected a serine residue in the cm-

30

related region of RAP30 from phosphorylation in vitro. In addition, human RAP30fl4
bound to E. coli RNA polymerase and was displaced by o70. These results suggest that
RAP30 and o70 have functionally related RNA polymerase-binding regions and are able
to bind to the same region of RNA polymerase (McCracken and Greenblatt, 1991).
RAP30 alone can prevent RNAP II from binding nonspecifically to DNA and greatly
inhibit nonspecific transcription by RNAP II (Killeen and Greenblatt, 1992).
Furthermore, recombinant RAP30 is sufficient for recruitment of RNA polymerase II to
the DAB complex (Flores et alt, 1991; Killeen et al., 1992). This ability of RAP30 to
recruit RNA polymerase to a promoter is also a characteristic of o factors in prokaryotes.
The findings that the RAP30 C-terminus is a cryptic DNA-binding domain (Tan et al.,
1994) and that RAP30 can be crosslinked in the pre-initiation complex to promoter
sequences between the TATA box and the transcription start site (Coulombe et al., 1994)
- suggest that TFIIF stabilizes the pre-initiation complex by nonspecifically binding to
promoter DNA through its RAP30 subunit. This region of the promoter between the
TATA box and Initiator is not known to contain specific promoter elements.
Recombinant RAP30 by itself cannot release RNAP II from non-promoter DNA
once it has bound, although RAP30fl4 has this capacity (Killeen et al., 1992; Killeen and
Greenblatt, 1992). Other studies also indicate that RAP74 participates in the interaction
of RAP30/74 with RNAP II by stabilizing either the interaction between RAP30 and
RNAP 11, between RNAP II and DNA, or between RAP30 and DNA.(Garrett et al., 1992;
Tyree et al., 1993; Coulombe et al., 1994). Indeed, RAP74 can directly interact with
RNAP 11 through its C-terminus, which is masked by its N-terminus and central region
(Wang and Burton, 1995). Although RAP30 can recruit RNAP II to the DAB complex
without RAP74 (Flores et al., 1991; Killeen et al., 1992), positioning of RAP30 close to
the promoter within the pro-initiation complex requires RAP74 (Coulombe et al., 1994).
RAP74 can enhance the binding of bacterial RNA polymerase to a promoter and
stimulates transcription in vitro by bacterial RNA polymerase (Chibazakura et al., 1994).

31

Evidence from both genetic and biochemical studies indicate that there may be
significant coupling of TFIIB and TFIIF function in transcription. Both TFIIB and TFIIF
are components of a minimal core transcription machinery (Tyree et al., 1993). The SUA7
gene in yeast encodes TFIIB, and the RAP74 homologue in yeast is encoded by
SSU71/TFGI . An ssu71 mutant supresses a cold-sensitive sua7 mutant, which alters the
initiation start site (Sun and Hampsey, 1995). RAP74 stimulates CTD phosphatase
activity, apparently through its interaction with RNAP II. TFIIB, however, suppresses
stimulation of phosphatase activity by RAP74 (Chambers et al., 1995). In addition,
RAP30 interacts physically with the N-terminal region of TFIIB (Ha et al., 1993; Chapter
III). Thus, TFIIF interacts genetically, biochemically, and physically with TFIIB. Both
RAP30 and RAP74 have also been shown to bind TFIIB (Maxon et al., 1994).

TFIIF also functions in activated transcription. A low amount of TFIIF was
sufficient for basal transcription, whereas a higher quantity of TFIIF was required for
activated transcription by serum response factor (SRF) and GAL4-VP16, but not Spl.
T'FIIF could relieve squelching by SRF in vitro, and RAP74 bound to DNA interacted
with either SRF or GAL4-VP16 (Zhu et al., 1994). Another study identified TFIIF as a
stoichiometric component of a multiprotein mediator complex that responds to both
GAL4-VP16 and GCN4 activators for transcription in vitro (Y .I . Kim et al., 1994).

In addition to being essential for initiation, RAP74 has also been implicated as
being required for promoter escape at least under some circumstances (Chang et al.,
1993). TFIIF also stimulates transcription elongation (Price et al., 1989). Mechanistic
studies suggest that TFIIF increases the overall rate of RNA chain elongation by RNAP II
by suppressing transient pausing of RNAP II at many sites on DNA templates (Flores et
al., 1989; Izban and Luse, 1992; Bradsher et al., 1993; Kephart et al., 1994; Tan et al,
1994). TFIIF stimulates the basal level of elongation, but not the activated level of
elongation by the HIV trans-acting protein Tat. Antiserum against RAP74 preferentially

suppresses activation by Tat, suggesting that Tat may simulate elongation through

32

RAP74 (Kato etal., 1992). It was proposed that the stimulation of elongation by TFIIF is
mediated by the phosphorylation of its RAP74 subunit (Kitajima et al., 1994). Other
studies suggest that both RAP30 and RAP7 4 function in stimulation of the elongation by
RNAP II (Tan et al., 1994; Tan et al., 1995).

33

OVERVIEW

Our laboratory has focused on functions of TFIIF, which has essential functions
during the initiation and elongation stages of the transcription cycle by RNA polymerase
II. A HeLa cell extract depleted of TFIIF has been used to assay TFIIF function in vitro
(Burton et al., 1986, 1988). Cloning of cDNAs encoding the RAP30 and RAP74 subunits
of TFIIF (Sopta et al., 1989; Finkelstein et al., 1992) and bacterial production of
recombinant RAP30 and RAP74 proteins (Wang et al., 1993, 1994) has greatly facilitated
further studies. A set of deletion mutants of RAP74 has also been constructed and
assayed (Wang and Burton, 1995).

Since RAP30 has sigma-like function, several research groups have tried to
identify the regions of RAP30 similar to the conserved regions of bacterial sigma factors
(Sopta et al., 1989; McCracken and Greenblatt, 1991; Gong et al., 1992; Garrett et al.,
1992). These previously reported sequence comparisons were based on linear alignment
methods, and may not be reliable. In Chapter II, we applied a 2-dimensional sequence
analysis method named Hydrophobic Cluster Analysis to compare amino acids sequences
of bacterial sigma factors, B. subtilis delta protein, human and Drosophila RAP30, and
yeast ng2p and Cdc7 3p. Our analysis indicates that RAP30 contains sequences similar
and co-linear with conserved regions of sigma factors. The N-terminal region of RAP30
is similar to subregion 1.2 of bacterial sigma factors. The central region of RAP30 has
been aligned to the core binding domain of sigma factors, subregion 2.1. The C-terminal
region of RAP30 is similar to subregions 3.1 and 4.1 which are implicated in DNA
binding. The C-terminal end of RAP30 may have similarity to the N-terminal region of
delta protein. These results suggest that human RAP30 is structurally related to sigma and
delta factors, RNA polymerase binding proteins from bacteria. Based on the sequence
comparison, we suggest that yeast Cdc73p may be similar to RAP30, sigma and delta

34

factors. Cdc73p may be a subunit of an alternative version of TFIIF in yeast. The work
described in this chapter is in preparation for publication.

In Chapter III, we report the experiments to map the functional domains of
RAP30. A set of deletion mutants of human RAP30 was constructed with 6 histidine tags
at the C-termini to facilitate the purification and binding reactions. The C-terminal
histidine tag causes freshly renatured RAP30 to behave as a monomer in gel filtration,
whereas the unmodified RAP30 is a dimer. Each of these mutants was tested for the
ability to stimulate accurate runoff transcription from the Adenovirus major late
promoter. Transcription activity of RAP30 was very sensitive to deletion of N-terminal or
C-terminal sequences, indicating the importance of both N -and C-terminal sequences for
RAP30 function. The RAP74 binding site on RAP30 was mapped by immobilizing
histidine-tagged RAP30 mutants on nickel-chelate resin and testing the ability of this
affinity matrix to retain RAP74. The results indicate that RAP74 binds to the N-terminal
region of RAP30 between amino acids 1-98. The surfaces of RAP30 that interact with
RNA polymerase II were determined using affinity beads containing covalently
immobilized RNA polymerase II. The results demonstrate that RAP30 N -terminal
sequences between 1-50 and central sequences between 131-159 and 152—176 are
important for polymerase binding. These results are consistent with the sequence analysis
described in Chapter II and indicate the significance of our RAP30 alignments with sigma
subregions 1.2 and 2.1. The region ofRAP30 that binds toTFlIB was mappedusing two
methods that may differ in the sensitivity of detection of protein-protein interactions.
Using an affinity bead procedure in which TFIIB was covalently immobilized, an
extensive surface of RAP30, localized to amino acids 1-176, was found to contribute to
TFIIB binding. A minimal interaction domain was located between amino acids 27-118
by a more sensitive ELISA assay. Apparently, multiple contacts within the RAP74-
binding and RNA polymerase II-binding regions of RAP30 contribute to TFIIB binding.
It has been reported that deletion of the N-terminal region of RAP30 activates the DNA-

35

binding activity of the C-terminal region (Tan et al., 1994). We demonstrate here that
deletion of the N-terminal region of RAP30 reduces RAP74, RNA polymerase II and
TFIIB binding. The N-terminal region of RAP30, therefore, might regulate the function
of central and C-terminal domains. Part of the work described in this chapter has been
submitted to the Journal of Biological Chemistry for publication.

There are several lines of evidence indicating that there may be significant
coupling of TFIIF and TFIIB function in transcription (Ha et al., 1993; Sun and
Hampsey, 1995; Chambers et al., 1995). In Chapter IV, we investigated the relationship
between TFIIF and TFIIB. Using an affinity bead procedure in which TFIIB was
immobilized, TFIIB is shown to bind directly and independently to both RAP30 and
RAP74. This result confirms the previous report of a direct interaction between TFIIB
and RAP30 (Ha et al., 1993) and demonstrates for the first time that TFIIB also interacts
directly with RAP74. A set of RAP74 mutants was tested for binding to TFIIB. The
TFIIB binding site on RAP74 appears to be located within the C-terminal region between
amino acids 358-517, and is masked by the N-terminal region and central region.
Sequences within the N-terminal region and central region of RAP74 mask the C-
terminal region for RNA polymerase II binding (Wang and Burton, 1995) and CID
phosphatase stimulation (Chambers et al., 1995). Using an affinity bead procedure and
ELISA assays, we found that RAP74 antagonizes the interaction between TFIIB and
RAP30 through two mechanisms. By binding the N-terminal region of RAP30, the N-
terminal region of RAP74 can block interaction between RAP30 and TFIIB. Also, by
binding TFIIB, the C-terminal region of RAP74 can block this interaction. RAP30,
therefore, binds to TFIIB only in the absence of the RAP74 subunit. When the TFIIF
complex is intact, TFIIF-TFIIB contact is most likely maintained through the C-terminal
region of RAP74. We present three models to describe alternate pathways for initiation
and productive elongation based on dynamic interaction between TFIIB and TFIIF

subunits. The dynamic interaCtion between TFIIB and TFIIF subunits may be a

36

mechanism to separate RAP30 and RAP74 functions during various stages of the
transcription cycle. The importance of dynamic interactions between RAP30 and RAP74
subunits of TFIIF is indicated by the work of Chang et al. (1993) in which it was shown
that RAP30 and RAP74 have partially separable activities in accurate initiation and early
elongation. Work described in Chapter IV has been submitted to the Journal of Biological
Chemistry for publication.

37

REFERENCES

Allison, L.A., and Ingles, CJ. (1989). Proc. Natl. Acad. Sci. USA 86, 2794-2798.
Allison, L.A., Moyle, M., Shales, M., and Ingles, CJ. (1985). Cell 42, 599-610.

Allison, L.A., Won , J.K.-C., Fitzpatrick, V.D., Moyle, M., and Ingles, CJ. (1988). Mol.
Cell. Biol. 8, 321-3 9.

Aso, T., Vasavada, H.A., Kawaguchi, T., Germino, F.J., Ganguly. S., Kitajima, S.,
Weissman, S. M., and Yasukochi, Y. (1992). Nature 355, 461-464.

Auble, D.T., and Hahn, S. (1993). Genes Dev. 7, 844-856.

Bagby, S., Kim, S., Maldonado, E., Tong, K.I., Reinberg, D., and Ikura, M. (1995). Cell
82. 857-867.

Barberis, A., Muller, C.W., Harrison, SC, and Ptashne, M. (1993). Proc. Natl. Acad. Sci.
USA 90, 5628-5632.

Béagglgnei, M.S., Halden, N.F., Cullen, CR, and Corden, IL. (1988). Mol. Cell. Biol. 8,
3 .

Bengal, E., Flores, 0., Krauskopf, A., Reinberg, D., and Aloni, Y. (1991). Mol. Cell.
Biol. 11, 1195-1206.

Bernstein, R., DeJong, J., and Roeder, R.G. (1994). J. Biol. Chem. 269, 24361-24366.
Berroteran, R.W., Ware, D.E., Hampsey, M. (1994). Mol. Cell. Biol. 14, 226-237.

Bradsher, J.N., Tan, 8., McLaury, H.-J., Conaway, J.W., and Conaway, RC. (1993). J.
Biol. Chem. 268, 25594-25603.

Bradsher, J.N., Jackson, K.W., Conaway, R.C., and Conaway, J.W. (1993b). J. Biol.
Chem. 268, 25587-25593.

Breathnach, a, and Chambon, P. (1981). Ann. Rev. Biochem. 50, 349-383.

Bunick, D., Zandomeni, a, Ackerman, s., and Weinmann, R. (1982). Cell 29, 877-886.
Buratowski, s. (1994). Cell 77, 1-3.

Buratowski, s., and Sharp, RA. (1990). Mol. Cel. Biol. 10, 5562-5564.

Buratowski, S., and Zhou, H. (1992). Science 255, 1130-1132.

Buratowski, s., and Zhou, H. (1993). Proc. Natl. Acad. Sci. USA 90, 5633-5637.
Buratowski, s., Hahn, s., Guarent, L., and Sharp, RA. (1989). Cell 56, 549-561.

Buratowski, S., Sogta, M., Greenblatt, J., and Sharp, RA. (1991). Proc. Natl. Acad. Sci.
USA 88, 7509-751 . .

38

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

Burton, Z.F., Killeen, M., Sopta, M., Ortolan, L.G., and Greenblatt, J. (1988). Mol. Cell.
Biol. 8, 1602-1613.

Cadena, D.L., and Dahmus, M.E. (1987). J. Biol. Chem. 262, 12468-12474.

Carles, C., Triech, I., Bouet, F., Riva, M., and Sentenac, A. (1991). J. Biol. Chem. 266,
24092-24096.

Cavallini, B., Fans, 1., Matthes, H., Chipoulet, J.M., Winsor, B., Egly, J.M., and
Chambon, P. (1989). Proc. Natl. Acad. Sci. USA 86, 9803-9807.

Chambers, R.S., and Dahmus, ME. (1994). J. Biol. Chem. 269, 26243-26248.

Chambers, R.S., Wang, B.Q., Burton, Z.F., and Dahmus, M.E. (1995). J. Biol. Chem.
270, 14962-14969.

Chang, C., Kostrub, C.F., and Burton, Z.F. (1993). J. Biol. Chem. 268, 20482-20489.

Chen, J.-L., Attardi, L., Verrijzer, C.F., Yokomori, K., and Tjian, R. (1994). Cell 79, 93-
105.

Chiang, C.-M., Ge, H., Wang, Z., Hoffmann, A., and Roeder, R.G. (1993). EMBO J. 12,
2749-2762.

Chibazakura, T., Kitajima, S., Yonaha, M., and Yasukochi, Y. (1994). Biochimica et
Biophysica Acta 1219, 592-600.

Colgan, J ., Ashali, H., and Manley, J. (1995). Mol. Cell. Biol. 15, 2311-2320.

Comai, L., Tanese, N., and Tjian, R. (1992). Cell 68, 965-976.

Conaway, J.W., and Conaway, R.C. (1989). J. Biol. Chem. 264, 2357-2362.

Conaway, J.W., and Conaway, R.C. (1990). Science 248, 1550-1553.

Conaway, R.C., and Conaway, J .W. (1993). Annu. Rev. Biochem. 62, 161-190.
Conaway, J .W., Travis, B., and Conaway, R.C. (1990). J. Biol. Chem. 265, 7564-7596.

Egaway, R.C., Bradsher, J.N., and Conaway, J.W. (1992). J. Biol. Chem. 267, 8464-
7. -

Camagvay, R.C., Bradsher, J.N., and Conaway, J.W. (1992b). J. Biol. Chem. 267, 10142-
1 14 .

Cormack, B.P., and Struhl, K. (1992). Cell 69, 685-696.
Cormack, B.P., Strubin, M., Ponticelli, A.S., and Struhl, K. (1991). Cell 65, 341-348.
Cortes, P., Flores, 0., and Reinberg, D. (1992). Mol. Cell. Biol. 12, 413-421.

39

Coulombe B., Li, J., and Greenblatt, J. (1994). J. Biol. Chem. 269, 19962-19967.

Croston, G.E., Kerrigan, L.A., Lira, L.M., Marshak, D.R., and Kadonaga, J.T. (1991).
Science 251, 643-649.

Dahmus, ME. (1994). Transcription Mechanisms and Regulation, ed. by Conaway, R.C.
and Conaway, J .W., New York: Raven Press, 243-262.

Davidson, B.L., Egly, J .-M., Mulvihill, E.R., and Chambon, P. (1983). Nature (London)
301. 680-686.

DeJong, and Roeder, R.G. (1993). Genes Dev. 7, 2220-2234.
Drapkin, R., and Reinberg, D. (1994). Trends Biochem. Sci. 19, 504-508.
Drapkin, R., Merino, A., and Reinberg, D. (1993). Curr. Opin. Cell Biol. 5, 469-476.

Drapkin, R., Reardon, J., Ansari, A., Huang, J.-C., Zawel, L., Ahn, K., Sancar, A., and
Reinberg, D. (1994). Nature 368, 769-772.

Dynlacht, B.D., Hoey, T., and Tjian, R. (1991). Cell 66, 563-576.
Feaver, W.J., Gileadi, 0., Li, Y., and Kornberg, 1w. (1991). Cell 67, 1223-1230.

Feaver, W.J., Svejstrup, J .Q., Bardwell, L., Bardwell, A.J., Buratowski, S., Gulyas, K.D.,
Donahue, T.F., Friedberg, R.C., and Konrberg, RD. (1993). Cell 75, 1379-1387.

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

Flanagan, P.M., Kelleher, RJ. 1]], Feaver, W.J., Lue, N.F., LaPointe, J .W., and Kornberg,
RD. (1990). J. Biol. Chem. 265, 11105-11107.

Flores., 0., Maldonado, B., Burtonz, Z.F., Greenblatt, J ., and Reinberg, D. (1988). J. Biol.
Chem. 263, 10812-10816.

Flores, 0., Maldonado, B., and Reinberg, D. (1989). J. Biol. Chem. 264, 8913-8921.
Flores, 0., Ha, I., and Reinberg, D. (1990). J. Biol. Chem. 265, 5629-5634.

Flores, 0.. Lu, H., Killeen, M.T., Greenblatt, J., Burton, 2.1—1, and Reinberg, D. (1991).
Proc. Natl. Acad. Sci. USA 88. 9999-10003.

Flores, 0., Lu, H., and Reinberg, D. (1992). J. Biol. Chem. 267, 2786-2793.

Frank, D.J., Tyree, C.M., George, C.F., and Kadonaga, J .T. (1995). J. Biol. Chem. 270,
6292-6297.

Garrett, K.P., Serizawa, l-L, Hanley, J.P., Bradsher, J .N ., Tsuboi, A., Ami, N., Yokom, T.,
Aral, K., Conaway, R.C., and Conaway, J .W. (1992). J. Biol. Chem. 267, 23942-23949.

Ge, H., and Roeder, R.G. (1994). Cell 78, 513-523.

40

Ge, H., Zhao, Y., Chait, B.T., and Roeder, R.G. (1994). Proc. Natl. Acad. Sci. USA 91,
12691-12695.
Gill, G., and Tjian, R. (1991). Cell 78, 513-523.

Gong, D.-W., Hashimoto, S., Wada, K., Roeder, R.G., Nakatani, Y., and Horikoshi, M.
(1992). Nucleic Acids Res. 20, 6414.

Gong, D.-W., Hasegawa, S., Wada, K., Roeder, R.G., Nakatani, Y., and Horikoshi, M.
(1992b). Nucleic Acids Res. 20, 6736.

Gong, D.-W., Mortin, M.A., Horikoshi, M., and Nakatani, Y. (1995). Nucleic Acids Res.
23, 1882-1886.

Goodrich, J.A., and Tjian, R. (1994). Cell 77, 145-156.

Goodrich, J.A., and Tjian, R. (1994b). Curr. Opin. Cell Biol. 6, 403-409.

Goodrich, J.A., Hoey, T., Thut, C., Admon, A., Tjian, R. (1993). Cell 75, 519-530.
Grosschedl, R., and Birnsteil, ML. (1980). Proc. Natl. Acad. Sci. USA 77, 1432-1436.

Guzder, S.N., Sung, P., Bailly, V., Prakash, L., and Prakash, S. (1994). Nature 369, 578-
381. .

Ha, 1., Lane, W.S., and Reinberg, D. (1991). Nature 352, 689-695.

Ha, L, Roberts, 8., Maldonado, B., Sun, X., Kim, L., Green, M., and Reinberg, D. (1993).
Genes Dev. 7, 1021-1032.

Hahn, S., Buratowski, 8, Sharp, P.A., and Guarente, L. (1989). Cell 58, 1173-1181.
Ham, J., Steger, G., and Yaniv, M. (1992). FEBS 307, 81-86.
Henry, N.L., Sayre, M.H., and Kornberg, RD. (1992). J. Biol. Chem. 267, 23388-23392.

Henry, N.L., Campbell, A.M., Feaver, W.J., Poon, D., Wei], P.A., and Kornberg, RD.
(1994). Genes Dev. 8. 2868-2878.

Hernandez, N. (1993). Genes Dev. 7, 1291-1308.
Hisatake, K., Roeder, R.G., and Horikoshi, M. (1993). Nature 363, 744-747.

Hloegy, T8,6Dynlacht, B.D., Peterson, M.G., Pugh, B.P., and Tjian, R. (1990). Cell 61,
1 7 ~11 .

Hoey, T., Weinzierl, R., Gill, G., Chen, J.-L., Dynlacht, B.D., and Tjian, R. (1993). Cell
72, 247-260.

Hoffmann, A., Sinn, B., Yamamoto, T., Wang, J., Roy, A., Horikoshi, M., and Roeder,
R.G. (1990). Nature 346, 387-390.

Hoopes, B.C., LeBlanc, J.P., and Hawley, D.K. (1992). J. Biol. Chem. 267, 11539-11547.

41
Horikoshi, M., Wan , C.K., Fujii, H., Cromlish, J.A., Weil, P.A., and Roeder, R.G.
(1989). Nature 341, 2 -303.

Horikoshi, M., Yamamoto, T., Ohkuma, Y., Weil, P.A., and Roeder, R.G. (1990). Cell
61, 1171-1178.

Horikoshi, M., Bertuccioli, C., Takada, R., Wang, J., Yamamoto, T., and Roeder, R.
(1992). Proc. Natl. Acad. Sci. USA 89, 1060-1064.

Huet, J ., and Sentenac, A. (1992). Nucleic Acids Res. 20, 6451-6454.

Humbert, 8., Van Vuuren, H., Lutz, Y., Hoeikmakers, J.H., Egly, J.M., and Mancollin, V.
(1994). EMBO J. 13, 2393-2398.

Imbalzano, A.N., Kwon, H., Green, M.R., and Kingston, RE. (1994). Nature (London)
370, 481-485.

Ingles, C.J., Shales, M., Cress, W.D., Triezenberg, S., and Greenblatt, J. (1991). Nature
351. 588-590.

Inostroza, J., Flores, 0., and Reinberg, D. (1991). J. Biol. Chem. 266, 9304-9308.
Izban, M.G., and Luse, D.S. (1992). J. Biol. Chem. 267, 13647-13655.

Jacob, G.A., Luse, S.W., and Luse, D.S. (1991). J. Biol. Chem. 266, 22537-22544.
Jacob, G.A., Kitzmiller, J.A., and Luse, D.S. (1994). J. Biol. Chem. 269, 3655-3663.

13$, X., Brou, C., Lutz, Y., Davidson, 1., Chambon, P., and Tom, L. (1994). Cell 79,
-117.

Kane, C.M.(l994). Transcription Mechanisms and Regulation, ed. by Conaway, R.C. and
Conaway, J .W., New York: Raven Press, 279-296.

Kao, C.C., Lieberman, P.M., Schmidt, M.G., Zhou, Q., Pei, R., and Berk, A.J. (1990).
Science 248, 1646-1650.

Karlin, S. (1993). Proc. Natl. Acad. Sci. USA 90, 5593-5597.

Kassavetis, G.A., Joazeiro, C.A.P., Pisallo, M., Geiduschek, B.P., Colbert, T., Hahn, S.,
and Blanca. J .A. (1992). Cell 71, 1055-1064.

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

Kephart, D.D., Price, M.P., Burton, Z.F., Finkelstein, A., Greenblatt, J ., and Price, DH.
(1993). Nucleic Acids Res. 21, 1319.

Kephart, D.D., Wang, B.Q., Burton, Z.F., and Price, D.H. (1994). J. Biol. Chem. 269,
13536-13543.

Kerppola, T.K. and Kane, CM. (1988). Mol. Cell. Biol. 8, 4389-4394.
KCTPPOIE. T.K., and Kane, CM. (1990). Biochemistry 29, 269-278.

42

Kerppola. T.K., and Kane CM. (1991). FASBB J. 5, 2833-2842.

Khoury, G., and Gruss, P. (1983). Cell 33, 313-314.

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

Killeen, M., Coulombe, B., and Greenblatt, J. (1992). J. Biol. Chem. 267, 9463-9466.
Kim, J.L., Nikolov, DB, and Berley, SK. (1993). Nature 365, 520-552.

Kim, T.K., and Roeder, R.G. (1994). Proc. Natl. Acad. Sci. USA 91, 4170-4147.

Kim, W.Y., and Dahmus, ME. (1988). J. Biol. Chem. 264, 11511-11520.

Kim, W.Y., and Dahmus,.ME. (1989). J. Biol. Chem. 264, 3169-3176.

Kim, Y., Geiger, J.H., Hahn, S., and Sigler, RB. (1993). Nature 365, 512-519.

Kim, Y-J, Bjorklund, S., Li, Y., Sayre, M., and Kornberg, R. (1994). Cell 77, 599-608.

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

Kitajima, S., Chibazakura, T., Yonoha, M., Yasukochi, Y. (1994). J. Biol. Chem. 269,
29970-29977.

Kobayashi, Y., Kitajima, S., and Yasukochi, Y. (1992). Nucleic Acids Res. 20, 1994.
Koleske, AJ., and Young, R.A. (1994). Nature (London) 368, 466-469.

Koleske, AJ., and Young, R.A. (1995). Trends Biochem. Sci. 20, 113-116.
Kolodziej, P.A., and Young, R.A. (1991). Mol. Cell. Biol. 11, 4669-4678.

Kolodziej, P.A., Woychik, N.A., Liao, S.-M., and Young, R.A. (1990). Mol. Cell. Biol.
10, 1915-1920.

Kretzschmar, M., Meisterernst, M., and Roeder, R.G. (1993). Proc. Natl. Acad. Sci USA
90, 11508-11512.

Kretzschmar, M. Kaise, K., Louspeich, P., and Meisteremst, M. (1994). Cell 78, 525-534.

Kwon, H., Imbalzano, A.N., Khavari, P.A., Kingston, RE, and Green, MR. (1994).
Nature (London) 370, 477-481).

Laybourn, P.J., and Dahmus, ME. (1989). J. Biol. Chem. 264, 6693-6698.
Layboum, P.J., and Dahmus, ME. (1990). J. Biol. Chem. 265, 13165-13173.
Lee, D.K., Horikoshi, M., and Roeder, RD. (1991). Cell 67, 1241-1250.

Lee, D.K., Dejong, J., Hashimoto, S., Horikoshi, M., and Roeder, R.G. (1992). Mol. Cell.
Biol. 12, 5189-5196.

43

Lewin, B. (1990). Cell 61, 1161-1164.

Li, Y., Flanagan, P.M., Tschochner, H., Kornberg, RD. (1994). Science 263, 805-807.
Lin, Y.S., and Green, MR (1991) Cell 64, 971-981.

Linn, S.C., and Luse, D.S. (1991). Mol. Cell. Biol. 11, 1508-1522

Lobo, S.M., Tanaka, M., Sullivan, M.L., and Hernandez, N. (1992). Cell 71, 1029-1040.

Lu, H. Flores, 0., Weinmann, R., and Reinberg, D. (1991). Proc. Natl. Acad. Sci. USA
88, 10004-10008.

Lu, H., Zawel, L., Fisher, L., Egly, J.-M., and Reinberg, D. (1992). Nature 358, 641-645.
Luse, D.S., and Jacob, G.A. (1987). J. Biol. Chem. 262, 14990-14997.

Ma, D., Watanabe, H., Mermelstein, F., Admon, A., Oguri, K., Sun, X., Wada, T., Imai,
T., Shiroya, T., Reinberg, D., and Handa, H. (1993). Genes Dev. 7, 2246-2257.

Maldonado, B., Ha, J., Cortes, P., Weis, L., and Reinberg, D. (1990). Mol. Cell. Biol. 10,
6335-6347.

Maldonado. B. and Reinberg, D. (1995). Curr. Opio. Cell Biol. 7, 352-361.
Malik, S., Lee, D.K., and Roeder, R.G. (1993). Mol. Cell. Biol. 13, 6253-6259.
Maniatis, T., Goodbourn, S., and Fischer, J.A. (1987). Science 236, 1237-1244.

Manley, J.L., Fire, A., Cano, A., Sharp, P.A., and Gefter, M. (1980). Proc. Natl. Acad.
USA 87. 3624-3628.

Mann, C., Buhler, J.M., Treich, I., and Sentenac, A. (1987). Cell 48, 627-637.
Marshall N.F., and Price, DH. (1995). J. Biol. Chem. 270, 12335-12338.

W932i, T., Segall, J., Weil, A.P., and Roeder, R.G. (1980). J. Biol. Chem. 255, 11992-

Maxon, M.R., Goodrich, J .A., and Tjian, R. (1994). Genes Dev. 8, 515-524.
McCracken, S., and Greenblatt, J. (1991). Science 253. 900-902. '
McKnight, 5.1... and Kingbury, R. (1982). Science 217. 616-624.

Merino, A., Madden, K.R., Lane, W.S., Champoux, LL, and Reinberg, D. (1993). Nature
(London) 365, 227-232.

Mitchell, P.J., and Tjian, R. (1989). Science 245, 371-378.

Mancollin, V., Ro , R.D.W., and Bgl , J.-M. (1994). Transcription Mechanisms and
Regulation, ed. by naway, R.C. and naway, J .W., New York: Raven Press, 45-62.

44

Muhich, M.L., Lida, c.r., Horikoshi, M., Roeder, R.G., and Parker, cs. (1990). Proc.
Natl. Acad. Sci. USA 87, 9148-9152.

Myers, R.M., Tilly, K., and Maniatis, T. (1986). Science 232, 613-618.

Na, J.G., and Hampsey, M. (1993)., Nucleic Acids Res. 21, 3413-3417.

Nakatani, Y. Horikoshi, M., Brenner, M., Yamamoto, T., Besnard, F., Roeder, R.G., and
Freese, B. (1990). Nature (London) 348, 86-88.

Nikolove, D.B., Hu, S.H., Lin, J ., Gasch, A., Hoffmann, A., Horikoshi, M., Chua, N-H.,
Roeder, R.G., and Burley, S.K. (1992). Nature 360, 40—46.

Nikolove, D.B., Chen, H., Halay, B.D., Usheva, A.A., Hisatake, K., Lee, D.K., Roeder,
R.G., and Burley, S.K. (1995). Nature 377, 119-128.

Nonet, M., Sweetser, D., and Young, R.A. (1987). Cell 50, 909-915.
Ohkuma, Y. and Roeder, R.G. (1994). Nature 368, 160-163.

Ohkuma, Y., Sumimoto, H., Horikoshi, M., and Roeder, R.G. (1990). Proc. Natl. Acad.
Sci. USA 87. 9163-9167.

Ohkuma, Y., Sumimoto, H., Hoffmann, A., Shimasalci, S., Horikoshi, M., and Roeder,
R.G. (1991). Nature 354, 398-401.

Ohkuma, Y., Hashimoto, S., Wang, C.K., Horikoshi, M., and Roeder, R.G. (1995). Mol.
Cell. Biol. 15, 4856-4866.

Ozer, J., Moore, P.A., Bolden, A.H., Lee, A., Rosen, G.A., Lieberman, P. (1994). Genes
Dev. 8, 2324-2335.

Parker, C.S., and Topol, J. (1984). Cell 36, 357

P333, J.M., Laybourn, P.J., and Dahmus, ME. (1989). J. Biol. Chem. 264, 19621-
1 .

lz’ggangpe, S.M. Kamakaka, R.T., and Kadonaga, J .T. (1994). Annu. Rev. Biochem. 63,
2 .

Parvin. 1.1).. and Sharp, RA. (1993). Cell 76, 1115-1121.
Peterson, C.L., Kruger, W., and Herskowitz, I. (1991). Cell 64, 1135-1143.
Peterson, M.G., Tanese, N., Pugh, B.F., and Tjian, R. (1990). Science 248, 1625-1630.

Peterson, M.G., Inostroza, J., Maxon, M.R., Flores, 0., Admon, A., Reinberg, D., and
Tjian, R. (1991). Nature 354, 369-373.

Pinto, 1., Ware, D.B., Hampsey, M. (1992) Cell 68, 977-988.

grantoé 1., Wu, W.-H., Na, J.G., and Hampsey, M. (1994). J. Biol. Chem. 269, 30569-
57 .

45

Price, D.H., Sluder, A.E., and Greenleaf, AL. (1989). Mol. Cell. Biol. 9, 1465-1475.
Proudfoot, NJ. (1989). Trends Biochem. 14, 105-110.

Ptashne, M. (1988). Nature (London) 335, 683-689.

Ptashne, M., and Gann., A.A.F. (1990). Nature (London) 346, 329-331.

Pugh, B.P., and Tjian, R. (1990). Cell 61, 1187-1197.

Pugh, B.P., and Tjian, R. (1991). Genes Dev. 5, 1935-1945.

Ranish, J.A., Lane, W.S., and Hahn, S (1992). Science 255, 1127-1129.

Reese, J., Apone, L., Walker, 8.8., Griffin, L.A., and Green, MR. (1994). Nature 371,
523-527.

Reinberg, D., and Roeder, R.G. (1987). J. Biol. Chem. 262, 3310-3321.
Reinberg, D., Horikoshi, M., and Roeder, R.G. (1987). J. Biol. Chem. 262, 3322-3330.

laleines, D., Wells, D., Chamberlin, ML, and Kane, GM. (1987). J. Mol. Biol. 196, 299-
12.

Reines, D. (1994). Transcription Mechanisms and Regulation, ed. by Conaway, R.C. and
Conaway, J .W., New York: Raven Press, 263-278.

Rigby, P.W.J. (1993). Cell 72, 7-10.
Roberts, S., and Green, MR. (1994). Nature 371, 717-720.

Rabat; S., Hal, 1., Maldonado, B., Reinberg, D., and Green, M. (1993). Nature 363, 741-
7 .

Rudd, M.D., Izban, M.G., Luse, D.S. (1994). Proc. Natl. Acad. Sci. USA 91, 8057-8061.
Samuels, M.A., Fire, A., and Sharp, RA. (1982). J. Biol. Chem. 257, 14419- 14427.
Sawadogo. M., and Roeder, R.G. (1984). J. Biol. Chem. 257, 14419-14427.

3334:, M.H., Tschochner, H., and Kornberg, RD. (1992). J. Biol. Chem. 267, 23383-
7.

Scafe, C., Chao, D., Lopes, J., Hirsch, J.P., Henry, S., and Young, R.A. (1990). Nature
347, 491-494.

Schaeffcr, L., Roy, R., Humbert, S., Mancollin, V., Vermeulen, W., Hoeijmakers, J.H.,
Chambon, P., Egly, J .-M. (1993). Science 260, 58-63.

Schaeffer, L., Mancollin, V., Roy, R., Staub, A., Mezzina, M., Sarasin, A., Weeda, G.,
Hoeijimaker, J .H.J., and Egly. J.-M. (1994). EMBO J. 13, 2388-2392.

Schmide, M.C., Kao, C.C., Pei, R., and Berk, A.J. (1989). Proc. Natl. Acad. Sci. USA 86,
7785-7789.

Schult2. M.Z.. Reeder. RH, and Hahn, S. (1992). Cell 69, 697-702.

Serizawa, H., Conaway, R.C., and Conaway, J .W. (1992). Proc. Natl. Acad. Sci. USA 89,
7476-7480.

Serizawa, H., Conaway, J.W., and Conaway, R.C. (1994). J. Biol. Chem. 269, 20750-
20756.

Serizawa, H., Conaway, J.W., and Conaway, R.C. (1994b). Transcription: Mechanisms
ar3ld Regulation, ed. by Conaway, R.C. and Conaway, J .W., New York: Raven Press, 27 -
4 .

Serizawa, H., Makela, T.F., Conaway, J.W., Conaway, R.C., Weinberg, R.A., and Young,
R.A. (1995). Nature 374, 280-282.

Shiekhattar, R., Mermelstein, F., Fisher, R.P., Drapkin, R., Dynlacht, B., Wessling, H.C.,
Morgan, D.O., and Reinberg, D. (1995). Nature 374, 283-287.

Simmen, K.A., Bemues, H., Lewis, J.D., and Mattaj, J.W. (1992). Nucleis Acids Res. 20,
5889-5898.

Smale, S.T., and Baltimore, D. (1989). Cell 57, 103-113.

Smale, S.T., Schmidt, M.C., Berk, A.J., and Baltimore, D. (1990). Proc. Natl. Acad. Sci.
USA 87, 5268-5272.

Sopta, M., Burton, Z.F., and Greenblatt, J. (1989). Nature 341, 410-414.
Starr, D.B., and Hawley, D.K. (1991). Cell 67, 1231-1240.
Stringer, K.F., Ingles, CL, and Greenblatt, J. (1990). Nature 345, 783-786.

Sumimoto, H., Ohkuma, Y., Sinn, B., Kato, H., Shimasaki, S., Horikoshi, M., and
Roeder, R.G. (1991). Nature 354, 401-404.

Sun, Z.W., and Hampsey, M. (1995). Proc. Natl. Acad. Sci. USA 92, 3127-3131.

gaunéxa M11, D., Sheldon, M., Yeung, K., and Reinberg, D. (1994). Genes Dev. 8, 2336-

Sung, P., Higgins, D., Prakash, L., and Prakash, S. (1988). EMBO J. 7, 3263-3269.
Suzuki, M. (1990). Nature 344, 562-565.

Svejstr'up, J.Q., Wang, Z., Feaver, W.J., Wu, X., Bushnell, D.A., Donahue, T.F.,
Friedberg, B.C., and Kornberg, RD. (1995). Cell 80, 21-28.

Sgeéetser, D., Nonet, M., and Young, R.A. (1987). Proc. Natl. Acad. Sci. USA 84, 1192-

13188311- A.K.P., Fisher, T.S., and Pugh, B.F. (1992). Cell 71, 1015-1028.

47
Tan, S., Garrett, K.P., Conaway, R.C., and Conaway, J.W. (1994). Proc. Natl. Acad. Sci.
USA 91. 9808-9812.

Tan, S., Conaway, R.C., and Conaway, J.W. (1995). Proc. Natl. Acad. Sci. USA 92,
6042-6046.

Tantravahi, J., Alvira, M., and Falck-Pedersen, E. (1993). Mol. Cell. Biol. 13, 578-587.
Treich, 1., Charles, C., Riva, M., and Sentenac, A. (1992)., Gene Expr. 2, 31-37.
Tjian, R., and Maniatis, T. (1994). Cell 77, 5-8.

Tschochner, H., Sayre,lM.H., Flanagan, P.M., Feaver, W.J., and Kornberg, RD. (1992).
Proc. Natl. Acad. Sci. USA 89, 11291-11296.

Tyree, C.M., George, C.P., Lira-DeVito, L.M., Wampler, S.L., Dahmus, M.E., Zawel, L.,
and Kadonaga, J .T. (1993). Genes Dev. 7, 1254-1265.

Usheva, A., and Shenk, T. (1994). Cell 76, 1115-1121.

Usheva, A., Maldonado, B., Goldring, A., Lu, H., Houbavi, C., Reinberg, D., and Aloni,
Y. (1992). Cell 69, 871-881.

VanVuuren, A.J., Vermeulen, W., Ma, L., Weeda, G., Appeldoon, B., Jaspers, N.J.G.,
Var der Eb, A.J., Bootsma, D., Hoeijmakers, J .H.J ., Humbert, S. et 81. (1994). EMBO J.
13, 1645-1653. ,

Verrijzer, P., Yokomori, K., Chen, J.L., and Tjian, R. (1994). Science 264, 933-941.
Verlijzer, P., Chen, J-L., Yokomori, K., and Tjian, R. (1995). Cell 81, 1115-1125.
Wallle, B., and Keller, W. (1992). Annu. Rev. Biochem. 61, 419-440.

Wampler, S.L., and Kadonaga, J .T. (1992). Genes Dev. 6, 1542-1552.

Wang, B.Q., Kostrub, C.F., Finkelstein, A., and Burton, Z.F. (1993). Protein Expr. Purif.
4, 207-214.

Wang, B.Q., Lei, L., and Burton, Z.F. (1994). Protein Expr. Purif. 5, 476-485.
Wang, B.Q., and Burton, Z.F. (1995). J. Biol. Chem. 270, 27035-27044.
Wang, W., Carey, M., and Gralla, J .D. (1992). Science 255, 450-453.

Wang, Z., Svejstrup, J.Q., Feaver, W.J., Wu, X., Kornberg, RD, and Friedberg, EC.
(1994). Nature 368. 74-75.

Wasylyk, B., Derbyshire, R., Guy, A., et a1. (1980). Proc. Natl. Acad. Sci. USA 77, 7024-
7028.

Weil, P.A., Luse, D.S., Segall, J ., and Roeder, R.G. (1979). Cell 18, 469-484.
Weinmann, R. (1992). Gene Expression 2, 81-91.

48

Weis, L., and Reinberg, D. (1992). FASEB J. 6, 3300-3309.

Welch, M.D., and Drubin, D.G. (1994). Mol. Biol. Cell. 5, 617-632.

White, R.J., and Jackson, SP. (1992). Cell 71, 1041-1053.

Woychik, N.A., Lane, W.S., and Young, R.A. (1990). Genes Dev. 4, 313-323.

Woychik, N.A., and Young, R.A. (1994). Transcription: Mechanisms and Regulation, ed.
by Conaway, R.C. and Conaway, J .W., New York: Raven Press, 227-242.

Wu, D., Park, C.-H., Matsunaga, T., Hsu, D., Reardon, J.T., and Sancar, A. (1995). J.
Biol. Chem. 270, 2415-2418.

Xiao, H., Pearson, A., Coulombe, B., Truant, R., Zhang, S., Regier, J.L., Triezenberg,
S.J., Reinberg, D., Flores, 0., Ingles, Cl, and Greenblatt, J. (1994). Mol. Cell. Biol. 14,
7013-7023.

Yamamoto, T., Horikoshi, M., Wang, J. Hasegawa, S., Weil, P.A., and Roeder, R.G.
(1992). Proc. Natl. Acad. Sci. USA 89, 2844-2848.

Yamashita, S., Hisatake, K., Kobubo, T., Doi, K., Roeder, R.G., Horikoshi, M., and
Nakatani, Y. (1993). Science 261, 463-466.

Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D.L. (1994). Proc. Natl.
Acad. Sci. USA 91, 5237-5241.

Yokomori, K., Admon, A., Goodrich, J.A., Chen, J.-L., and Tjian, R. (1993). Genes Dev.
7, 2235-2245.

Yokomori, K., Zeidler, M.P., Chen, J .-L., Verrijzer, C.P., Mlodzik, M., and Tjian, R.
(1994). Genes Dev. 8, 2313-2323.

Yonaha, M., Aso, T., Kobayashi, Y., Vasavada, H., Yasukochi, Y., Weissman, S.M., and
Kitajima, S. (1992). Nucleic Acids Res. 21, 273-279.

Young, RA. (1991). Annu. Rev. Biochem. 60, 689-715.

Zawel, L., and Reinberg, D. (1993). Prog. Nucl. Acids Res. 44, 67-108.
Zawel, L., and Reinberg, D. (1995). Annu. Rev. Biochem. 64, 533-561.
Zawel, L., Kumar, K.P., and Reinberg, D. (1995). Gene. Dev. 9, 1479-1490.
Zehring, W.A., and Greenleaf, A.L. (1990). J. Biol. Chem. 265, 8351-8353.

Zehring, W.A., Lee, J .M., Weeks, J.R., Jokerst, R.S., and Greenleaf, A.L. (1988). Proc.
Natl. Acad. Sci. USA 85, 3698-3702.

Zhou, Q., Schmidt, M., and Berk, A. (1991). EMBO J. 10, 1843-1852.
Zhou, Q., Boyer, T.G., and Berk, A.J. (1993). Gene Dev. 7, 180-187.
Zhu, H., Joliot, V., and Prywes, R. (1994). J. Biol. Chem. 269, 3489-3497.

CHAPTER II

SEQUENCE ANALYSIS OF RAP30

49

50

ABSTRACT

The RAP30 subunit of general transcription factor TFIIF has functions analogous
to the functions of bacterial sigma factors. Using a 2-dimensional sequence analysis
method, named Hydrophobic Cluster Analysis, RAP30 was found to be structurally
related to bacterial sigma factors. RAP30 was found to contain sequences weakly similar
and co-linear with conserved subregions 1.2, 2.1, 3.1, and 4.1 of sigma factors. RAP30
also has sequence similar to the N-terminal region of delta protein, a non-essential
subunit of B. sublilis RNA polymerase. These proposed alignments are different from
those reported previously based on linear alignment methods. We also found that the
overall structure of yeast Cdc73p is similar to RAP30, suggesting that Cdc73p may be an

alternative subunit of yeast TFIIF.

51

INTRODUCTION

Transcription in bacteria is catalyzed by a multisubunit RNA polymerase, «2133'.
This core polymerase is unable to recognize promoter sequences and does not accurately
initiate transcription from promoter sites in the absence of a 6 factor. Sigma factors are a
family of relatively small, dissociable subunits of RNA polymerase, which bind to the
core polymerase to form the holoenzyme. The holoenzyme then can recognize and bind
to a promoter, permitting transcription initiation from a Specific site. Each holoenzyme
Specifically recognizes a set of related promoter elements with a consensus sequence
recognized by the Sigma factor. After transcription initiation, the sigma factor is released
and the core polymerase elongates an RNA chain (Reviewed by Helmann and
Chamberlin, 1988;He1mann, 1994).

Each Sigma factor has at least two functions: core binding and activation of
promoter recognition. Some sigma factors also have DNA-melting activity and inhibit
nonspecific transcription. The interaction of sigma factor with the core enzyme involves
sites on both the B and [3' subunits (Ishihama, 1992). Since there is a competition between
different sigma factors for core binding (21011 et al., 1992), it is likely that different sigma
factors bind to the same region of the core polymerase. Therefore, different sigma factors
may have similar core binding motifs. The binding of Sigma factor to core reduces the
affinity of the core for non-promoter DNA about 104 fold (Hinkle and Chamberlin,
1972). Genetic analyses indicate that Sigma factors play a direct role in determining
promoter recognition by contacting -35 and -10 consensus regions of promoter DNA
(Zuber et al., 1989; Siegele et a1, 1989; Gardella et al., 1989). Indeed, biochemical Studies
revealed two cryptic promoter DNA-binding domains of Sigma 70 (Dombroski et al.,
1992). '

52

Sigma factors can be divided into two unrelated groups: the sigma 70 family,
which consists of the primary and many of the alternative sigma factors, and the sigma 54
family. Sequence comparisons of members of the sigma 70 family shows that there are
four highly conserved regions, and some of these regions can be further divided into
subregions (Gribskov and Burgess, 1986; Helmann and Chamberlin, 1988; Lonetto et al.,
1992). Regions 2 and 4 are the most highly conserved regions, indicating that these two
regions are the most crucial for Sigma factor functions. Indeed, genetic and biochemical
Studies suggest that most of Sigma functions require portions of regions 2 and 4.
Subregion 2.1, the most highly conserved region, is involved in core binding (Lesley and
Burgess, 1989; Shuler et al., 1995). Subregion 2.3 is involved in DNA melting, which
helps to establish the open promoter complex (Jones and Moran, 1992; Jones et a1, 1992).
Subregion 2.4 recognizes the -10 region of the promoter (Siegele et al., 1989; Zuber et al.,
1989; Daniels et al., 1990; Waldurger et al., 1990; Dombroski et al., 1992), whereas
subregion 4.2, which contains a helix-tum-helix DNA-binding motif, interacts with the
-35 region of the promoter (Siegele et al., 1989; Gardella et al., 1989; Dombroski et al.,
1992). Regions 1 and 3 are missing in many members of the sigma 70 family. Region 1
masks both the promoter and nonpromoter DNA-bindin g domains of sigma 70
(Dombroski et al., 1992). The function of region 3 is unknown. It may be involved in
DNA-binding (Dombroski et al., 1992).

Bacillus subtilis RNA polymerase, in contrast to E. coli RNA polymerase,
contains an additional non-essential subunit, the 21 kDa 8 protein, which is encoded by
the mob? gene (Pero et al., 1975; Lampe et al.,.1988). Together with sigma, the delta
protein functions as an initiation subunit of polymerase to allow site-selective interaction
with DNA templates (Achberger and Whiteley, 1981). Both delta and sigma bind to core
Simultaneously but with negative cooperativity, and promoter recognition may involve a

ternary complex of E50 (Hyde et al., 1986). Delta inhibits transcription from non-specific
template (Tjian et al., 1977), reduces the affinity of RNA polymerase for nonpromoter

53

DNA (Dickel et al., 1980; Achberger and Whiteley, 1981), and decreases the association
of RNA polymerase with both promoter and non-promoter DNA (Achberger et al., 1982;
Hilton and Whiteley, 1985). Delta participates in both the initiation and core recycling
phases of the transcription cycle by inhibiting open complex formation at the promoter
and enhancing core enzyme recycling (J uang and Helmann, 1994).

The RAP30 subunit of general transcription factor TFIIF has Sigma-like and delta-
1ike functions. It can not only bind to eucaryotic RNAP II, but it can also compete with
sigma 70 to bind E. coli RNA polymerase (McCracken and Greenblatt, 1991). RAP30
can prevent RNAP II from binding nonspecifically to DNA and greatly inhibits
nonspecific transcription by RNAP II (Killeen and Greenblatt, 1992). Furthermore,
recombinant RAP30 is sufficient for recruitment of RNA polymerase II to the pre-
initiation complex (Flores et al., 1991; Killeen etal., 1992). Like sigma 70, RAP30 also
contains a cryptic DNA-binding domain at its C-temrinus (Tan et al., 1994). Therefore,
RAP30 appears to be a member of the Si gma family.

It was proposed that RAP30 binds to RNAP II through its central region which
has weak structural similarity to subregions 2.1 and 2.2 of sigma (McCracken and
Greenblatt, 1991). The C-terminus of RAP30 was also suggested to contain weak
similarity to subregion 4.2 of sigmas (Garrett et al., 1992). These sequence alignments,
using linear alignment methods, do not Show strong similarity and may be unreliable.
Here we use a 2-dimensional sequence analysis method, named Hydrophobic Cluster
Analysis (HCA), to compare amino acids sequences of sigma factors, delta protein,
human and Drosophila RAP30, and yeast Cdc73p. Cdc73p was previously described as
an extragenic suppressor of an (rt-factor receptor (STEZ) mutant (Reed et al., 1988) and
was recently identified as a novel yeast RNA polymerase II-associating protein (Wade et
al., 1995). HCA is a powerful method for comparing amino acids sequences of distinct
proteins, and can detect similarities in the 3D folding of proteins with very low sequence

identity (Gaboriaud et al., 1987; Lemesle-Varloot et al., 1990). HCA can also be used to

54

search homologous domains that are separated by variable segments (Henrissat et al.,
1988). Our analysis from HCA indicates that RAP30, as well as Cdc73p, may have
sequence similarities to conserved subregions 1.2, 2.1, 3.1, and 4.1 of sigma factors and
the N-terminal region of delta protein. These proposed alignments differ from those
reported previously based on linear alignment methods. Based on the sequence

comparison, we also suggest that Cdc73p may be an alternative subunit of yeast TFIIF.

55

METHODS

Hydrophobic Cluster Analysis

Amino acid sequences were obtained from GCG database. HCA plots were drawn
using a Macintosh plot program from Doriane S.A. (France). The amino acid sequence is
displayed as a classical a-helix projected in two dimensions and duplicated. Amino acids
are represented by the Standard one-letter code, except the following amino acids are
denoted by symbols: Proline by "Star", glycine by "diamond", cysteine by "c" with a
circle, threonine by "square", and serine by "square" filled with a spot. V, I, L, F, W, M,
and Y are considered hydrophobic. A and C can be considered hydrophobic in a
hydrophobic environment. Hydrophobic clusters composed of adjacent hydrophobic

residues that are not broken by prolines are circled.

Quality of Alignments

The quality of HCA alignments was calculated as the percentage of matched
amino acids after linear alignment following the indication from HCA. HCA alignment
scores were calculated as follows:

2CR x 100
HCA alignment score = --------------%
RC1 + RC2
Where RC1 (RC2) is the number of hydrophobic residues in cluster 1 (cluster 2). CR is
the number of hydrophobic residues in cluster 1 that are in correspondence with

hydrophobic residues in cluster 2 . If the HCA score is more than 60% along wide

segments of their sequences (more than 100 residues), 3D homologous proteins are

56

detected even if their sequence identity is as low as 10%. When the hydrophilic segments
localized between hydrophobic clusters are not conserved or differ in length, they can be
considered to correspond to loops and not to dramatically influence protein folding

(Gaboriaud et al., 1987).

57

RESULTS
1. Human RAP30 is structurally related to bacterial sigma factors

Hydmphobic Cluster Analysis revealed that human RAP30 contains several
regions weakly Similar to bacterial Sigma factors and delta protein. The N-terminus of
RAP30, which is the RAP74 binding domain, contributes to RNAP H binding (Chapter
III) and masks the DNA-binding C-terminus (Tan et al., 1994), is similar to subregion 1.2
of bacterial sigma factors, which is important for masking DNA-binding domains
(Dombroski et al., 1992) (Fig. 1). The central region of RAP30, which is involved in
RNAP II binding (McCracken and Greenblatt, 1991; Chapter H1), is similar to the core
binding domain of Sigma factors, subregion 2.1 (Lesley and Burgess, 1989; Shuler et al.,
1995) (Fig. 2). The cryptic DNA-binding C-terminus of RAP30 is Similar to subregions
3.1 and 4.1 of sigma factors, which are near DNA-binding subregions 2.4 and 4.2
(Dombroski et al., 1992) (Fig. 3). The C-terminal end of RAP30 has some Similarity with
the N-terminus of delta (Fig. 4).

2. The overall structure of yeast Cdc73p is similar to RAP30

The HCA plots of human RAP30, Drosophila RAP30 and yeast Cdc73p (the first
300 residues of 393) were compared (Fig. 5). The comparison of HCA plot of yeast
Cdc73p with HCA plots of RAP30S revealed a similar distribution of short hydrophobic
clusters over a length of about 300 residues, suggesting that Cdc73 might be structurally
related to RAP3OS. While the first 300 residues of Cdc73p only Share 13% and 11%
sequence identities with human RAP30 and Drosophila RAP30, respectively, their HCA
homology scores are as high as 69% and 66%, respectively (Table 1). If the HCA

58

homology scores are more than 60%, proteins are found to have the same overall 3D fold,
although their sequence identity may be as low as 10% (Gaboriaud et al., 1987).
Furthermore, like human RAP30, yeast Cdc73p contains sequences similar to subregions
1.2, 2.1, 3.1 and 4.1 of sigma factors and the N-terminal region of B. subtilis delta protein
(Fig. 1, 2, 3, 4). Notably, the Similarity between the C-terminus of Cdc73p and the N-
terminus of delta is more extensive than the Similarity between human RAP30 and delta
(Fig. 4).

In summary, based on HCA, both RAP30 and Cdc73p were found to contain
sequences similar and co-linear with conserved regions of sigma factors. RAP30 and

Cdc73p also have sequences similar to B. subtilis delta protein (Fig. 6).
3. Multiple sequence alignment of RAP30 and Cdc73p
A multiple sequence alignment of Cdc73p, a S. cerevisiae RAP30 homologue

ng2p (Henry et al., 1994), and Drosophila and human RAP30, based on HCA, is shown
in Fig. 7.

59

Figure l. The N-terminal regions of RAP30 and Cdc73p are similar to subregion 1.2
of bacterial sigma factors. HCA plots and deduced sequence alignments of N-terminal
regions of human RAP30, Drosophila RAP30 and S. cerevisiae Cdc73p, and subregion
1.2 of Sigma A (A na bena). Prosposed similar hydrophobic residues are Shaded.
Conserved amino acids are grouped as follow: (I, L, M, V, F, Y, W), (H, K, R), (D, E, N,
Q), (A, G), (S, T), and are underlined.

hRAP30 39
dRAP30 46
Cdc73p 36
Sigma A 88
(Anabaena)

ng2p 91

marine. . . . .BTaysrrmmaumarcsx '

WPG. . . .QKAQXSLSLTPAMLALDPEEKI
WETLSSDGSTQHSFPLNEETEIELDGSLVQLBIILHC
maximums. . . . .sKITLLmEmDSIanLE

|< >|
subregion 1.2

 

 

Ifignel

71
79

73

116

122

61

Figure 2. RNA polymerase II binding regions of RAP30 and Cdc73 are similar to
the core binding subregion 2.1 of bacterial sigma factors. HCA plots and deduced
sequence alignments of the central regions of human RAP30 and Drosophila RAP30, S.
cerevisiae Cdc73p, and subregion 2.1 of sigma A (Anabaena) and sigma 70 (E. coli).
Proposed Similar hydrophobic residues are Shaded. Bold indicates >50%, underline
indicates 30-50%, and italic indicates lO-30% match to a consensus sequence calculated
from 31 similar sigma factors. Stars indicate conserved residues between Cdc73p and
RAP30. The sequences of RAP30 and Cdc73p required for RNAP II binding are
indicated.

hRAP30
xRAP30
dRAP30

Cdc73p

>50%

30-50%

10-30%

(10%

142
156
158

131

62

required for pol II binding

 

SQQLDKVVTTNYKPVANHQYN.IEYERKKKEDGKR
SQQLEKAVTSNYXPVSNHQYN.IEYEKKKKDDGKR
VQPIDKIV.QNFKPVKDHAHN.IEYRERKKAEGKK

** ** ”k ** *‘k *‘k 'k *

VDIQNKTLAQELSTVKSTTSASLENDSEVSDPVVE

required for pol II binding

 

AK MVE NLRLVISIAKK.Y RGL F DLIQ

A INQL
B M E

riff-B

QE

3

RM SV RH V EGT LR S

L Y AVY S

SASAST ALVHRP AAK QN LPRGSEPQA RH

P DLA Q SN T E GS H SPNYKPS E
L T D Y KAAA

Figure2

176
190
192

165

 

63

Figure 3. The C-terminal region of RAP30 is Similar to subregions 3.1 and 4.1 of
bacterial sigma factors. HCA‘plotS and deduced sequence alignments of the C-terminal
region of human RAP30 and Drosophila RAP30, and subregions 3.1 and 4.1 of Sigma K
(B. subtilis). Prosposed similar hydrophobic residues are shaded. Conserved amino acids
are grouped as follows: (I, L, M, V, F, Y, W), (H, K, R), (D, E, N, Q), (A, G), (S, T), and
are underlined.

hRAP30
dRAP30

Cdc73p

Sigma K

hRAP30
dRAP30

Cdc73p

Sigma K

178
195

182

120

195
212

197

144

ADKQHELDMLESAFEKH....... 194
DDKNAMMDMLEHAFEKH....... 211

QAKPINEGXLIKQAE. . . . . . . . . 196
gumsummnzbvrbrrom 143

|<-—- subregion 3.1 --->|

QIYNLKDLEDIT--KQP¥¥ILKEILKEIGV
QYYNIKDLYKIT--NQPISXLKEILKD¥CD

o--~LKL¥QSIK*****!3NIKQELLESKS
ELEKMKQXIDILDDBEKEEIMGREGLDLKK

|< ------ subregion 4.1 -------- >|

*****: 46 a.a. deleted.

hRAP30

lfignedi

221
238

261

173

 

65

Figure 4. The C-terminal regions of RAP30 and Cdc73 are similar to the N-terminal
region of delta protein. HCA plots and deduced sequence alignments of the C-terminal
regions of human RAP30, Drosophila RAP30 and yeast Cdc73p, and the N-terminal
region of delta protein (B. subtilis). Prosposed similar hydrophobic residues are shaded.
Conserved amino acids are grouped as follows: (I, L, M, V, F, Y, W), (H, K, R), (D, E,
N, Q), (A, G), (S, T), and are underlined.

249
335
93

.....IflQENN 323

m w
l I
11‘"
N
x K
135-‘51
r
[‘0
dRAP30

...NTHBLKPEX...BHXQGEEKSD

223 QNEKGIHK...

hRAP30

...NMEELKKEX...BflXKTEEKKEEEHK 269

239 XNMKNPHK...

dRAP30

..§L¥H1EKNE...EBISR£1RF11VDN 296

MGIKQISQEELKEMALEIAHELFEEHKKPVEEQELLNEI 4 0

266 RNLPSXPH....
1

Cdc73p
Delta

299 TRME..TKPEXWDB!VAIEITGH......

41

Cdc73p

ASLLGVKKEELGDBIAQEXIDLNIDGRFLALSDQIKGLRS 80

Delta

324 IQ.flHSPQELEQR
81 flYPXDQLDEETQP

Cdc73p

Delta

 

hRAP30

Cdc73p

 

delta

Ifigumh4

67

Figure 5. HCA plots of human RAP30, DrOSOphila RAP30 and S. cerevisiae Cdc73p.

Vertical lines indicate the proposed regions of similarity between the segments.

 

68

Figure 5

 

69

Table 1

HCA alignment scores of hRAP30, dRAP30 and Cdc73p

 

Sequences hRAP30 dRAP30 Cdc73p

 

hRAP3O 100 80 69
(100) (50) (13)

dRAP30 100 66
(100) (11)

Cdc73p 100
(100)

 

For each entry, the HCA alignment score is given on top and the sequence identity score
(%) is given below in parentheses. The HCA alignment scores were calculated for each

cluster, using the following equation:

HCA alignment score = 2CR/(RC1 + RC2) x 100%

where RC1 and RC2 are the number of hydrophobic residues in cluster 1 and 2,
respectively. CR is number of hydrophobic residues in cluser 1 that are also found in
cluster 2. The mean value obtained for all the clusters along the sequences compared
gave the final HCA scores. HCA alignment scores of 60% are found among proteins

having the same overall fold although these proteins may have Significant structural

divergence.

70

Figure 6. The structure of human RAP30 and Cdc73p. The functional domains and

the regions similar to sigma factors and delta protein are indicated.

71

 

mmm _

 

o Ear

NF .

 

 

cog

ammo-go mo 9..»an

 

 

 

 

 

 

9.23.5

8.3. 5.5.. to 2.38

72

Figure 7. Multiple sequence alignment of human RAP30, Drosophila RAP30, S.
cerevisiae ng2p, and Cdc73p, based on HCA. Gaps are indicated by dots. Conserved
amino acids are grouped as follow: (I, L, M, V, F, Y, W), (H, K, R), (D, E, N, Q), (A, G),
(S, T), and are underlined.

.................................................. memo

................................................. mean

« exam 2: mascomuc>ac>H an Hm>amHamamunm Queue

.................................................... no

moH qu auaon

...... maymmqqoaumwummgumaaa«HaqmmeoH 22H a:uua on ouHoa aoH .......................................... omH annexe

..maaquammz: auzzuaaa ........... mcHHuH¢> HHn unpooo ............................................. nHH annexe

maH oHxsmmzHuumzzmmuaacanuuHmummuazmaaaamonumo «mH muons

mm HmawHuuum>uaqwmauzqquammamunuumunnueHuman: 4H auHoa HHH ........................... zmq>HHmumunauu. 6H annooo
up“ uawmuuxmmmmuuumamxmm.. .Huuuquaaz. pvu annexe

ova Gm 305.. .Hflg. «nu ong mm." Ragga. agamgm Nmn 435.3

m .......... Hauaaqmuquammuumuquu.. .Hummqanxu. eon muons ooH mucnumumwuu. amauammuuuzq. muuaumauxmammuaum onH ommamu

can gaiggma: .ga. new QMFOB 2.." 33. anggam mggfl mmmmm an

HnH nuqmuum..:an.unqzamau>nuzuuumu Hun... HuH Queue

«H mnqmuawxauuaz H ouHco moH uma.>mmnzuuw<m aaumaamnuuaqumzanau.. ..nam u ouH mnpooo
gga ooooooooooooo H0." 3%”

w ...; .noa :amqa H ............. uNN 0mg av." Hgflnfln Hgggnmggm no mg

.7. :88. 688888.. ............. .28. mm 833.8948 ................. ... ”m new.“
mom .... .gxmuuu. .:Hmau Ho gunman ............. mun muons

«bu ..... amumqummauumwmnquauuzmuyd HmmaamHHHHH on“ nmpooo nuH anaemanax.qummazumnxnuaa .................. noH unpooo

............................ . oHH mung nHH aoauHm

mm“ . ........................... «umzmmflgzauaaz "m “M 2: «5%. mg. 3% ........ g 2 83%

com . ........................... mam..HHauHauqz ooooo mama oOH Amaze mmmuamuaq. aaueu.. manuummuumam>m¢auucu no ommama

can .. .......................... mam..axuuqenqm an muons .......................................... a» ouH manna

« anmmeomm :ezau mHaemcexuummHumomHmenHmaaHuq. poH anpooo .......... amanuuuznqanzquoaauaas. .mmanzxxumqu on am one

Hauuzmuuamaaq «HH unnamuzmanuuu ......... unamHHmHmuHu om goauHm

auHa._H.m.$.§uusgaaw§ ........... 2 ”mm.“ 2. ogfiqmaafia:zu§z:aofi o. 8.2%

ha ....... gag? ............ H 8.33 3 gang ..... 326366.63 an 8.33

and ....... Huauquuqxaqummumzmu u mutumuu>n H manna oHH numHmnzazuquHHHuu ..... oouzumauuquaemnzzma no muons

ooooooooooooooo figHgoocooooooco FH QMPOB OD Hgmégaagmmflacccooa MM “GRUB

a ............................................ oaH annexe an unquHxn.muuuHuuum..enzmqaqauauuaauuuanumz H annexe

n: .......................................... n: omg an 3.3.5053 ...... mud: ....... H52

H H:zmz ammzmzuammza>u>au :QHHH H:muzzqn> Hammam> on manna «o mnuuenmz.mammuuuumzmmuHgaaqwmuuuuamzzuHnae «o auuua

h N N
oooooooooooooooooooooooooooooooooooooooooooooooo Rhos NM hagooooooggmaooee H “”8608

74

DISCUSSION

RAP30 can be considered a member of the sigma family. RAP30 binds to RNAP
II (McCracken and Greenblatt, 1991), recruits RNA polymerase II to the pre-initiation
complex (Flores et al., 1991; Killeen et al., 1992), prevents RNAP II from binding
nonspecifically to DNA, and greatly inhibits nonspecific transcription by RNAP II
(Killeen and Greenblatt, 1992). All these functions are analogous to the functions of
bacterial Sigma factors.

Several research groups have tried to identify the regions of RAP30 similar to
conserved regions of sigma factors. Sopta et al. proposed that human RAP30 contained a
region of Similarity with sigma 70. The best alignment was between residues 118-165 of
RAP30 and residues 373-425 of sigma 70, which corresponds to subregion 2.1 and 2.2.
The second-best alignment involved part of region 1b (residues 94-117 of RAP30 and
residues 111-139 of sigma 70) (Sopta et al., 1989). Since chopus RAP30 has an
insertion of 12 amino acids within the region of human RAP30 aligned to region 1b, this
alignment seemed unlikely to be correct (Gong et al., 1992). Garrett et al. proposed that
the C-terminus (residues ISO-249) of rat RAP30 was similar to regions 4.1 and 4.2 of
SpoIIIC (B. subtilis sigma k) (Garrett et al., 1992). The C-terminus of RAP30, however,
lacks the helix-turn-helix DNA-binding motif which is the primary feature of subregion
4.2. These previously reported sequence comparisons were based on linear alignment
methods, and may not be reliable.

HCA sometimes results in significant alignment in cases where linear methods
fail (Gaboriaud et al., 1987). Applying the HCA method, we have identified four sigma-
similar regions and one delta-similar region within the RAP30 sequence (Figs. 1, 2, 3, 4).
Notably, these regions are arranged in the same linear order in RAP30 as they are in

sigma factors, suggesting that RAP30 and Sigma factors might have similar structure.

75

The sequence similar to subregion 1.2 is near the N-terminus of RAP30. This is
the region that binds RAP74 and RNAP II (Chapter III) and masks DNA binding by the
RAP30 C-terminal region (Tan et al., 1994). This region of RAP30 is immediately distal
to a demonstrated RAP74-binding sequence that is highly conserved among RAP30 from
different species (Tan et al., 1995). Since subregion 1.2 of sigma factors masks DNA
binding domains, this region of RAP30 might be important for masking the C-terminal
DNA binding region. This region may also contribute to regulation of RAP74 and RNAP
II binding (Chapter III).

The central region of RAP30 was previously proposed to contain a subregion 2.1
similarity (Sopta et al., 1989), but our alignment, which is deduced from HCA, is
different from that which was previously proposed. The subregion 2.1 of Sigma factors is
the core binding domain (Lesley and Burgess, 1989; Shuler et al., 1995). There are
several lines of evidence indicating that the region of RAP30 that we have aligned to
subregion 2.1 may be involved in RNAP II binding. When RNAP II binds to RAP30, at
least one of the serine residues at positions 135, 136, or 142 is protected from
‘ phosphorylation (McCracken and Greenblatt, 1991). These serines residues are near the
aligned region. Mutagenic analysis suggests that the residues 131-159 and 152-176 of
RAP30, which are located in this region, are important for RNAP II binding (Chapter III).
A yeast Cdc73p mutant deleted 15 amino acids from this similar region decreases its
RNAP II binding affinity dramatically (C.-h. Chang and 2. F. Burton, unpublished data).
Thus, among these alignments, although the Similarity between RAP30 and sigma in this
region is weak, its functional role is the most clearly understood.

The C-terminal region of RAP30 includes a cryptic DNA binding domain (Tan et
al., 1994). This region contains sequences similar to subregions 3.1 and 4.1 of Sigma
factors and the N-terminal region of delta protein. Subregions 3.1 and 4.1 of sigma
factors may contribute to DNA binding by subregions 2.4 and 4.2 (Dombroski et al.,
1992). Although delta can limit RNAP binding to DNA, there is no direct evidence that

76

delta itself binds to DNA. Functional domains of delta have not yet been elucidated, so
the functional Significance of this similarity is unclear. It is possible that N-terminal
region of delta has DNA binding ability.

Like RAP30, sigma and delta, Cdc73p can be isolated by RNAP affinity
chromatography (Wade et al., 1995). Cdc73p appears to be evolutionarily related to
human RAP30, sigma factors and delta protein (Fig. 6). As expected, Cdc73p can bind to
Anclpfl‘fg3p, the smallest subunit of yeast TFIIF (L. Lei and Z. F. Burton, unpublished
data). Based on these observations, Cdc73p may be a homologue of yeast RAP30, but
this function has been ascribed to another yeast protein ng2p (Henry et al., 1994). This
raises the possibility that yeast, and possibly other eukaryotes, contains an alternative
form of RAP30, much as bacteria have alternative sigma factors for transcription of

different classes of genes.

77

REFERENCES

Achberger, B.C., and Whiteley, HR. (1981). J. Biol. Chem. 256, 7424-7432.

Achberger, B.C., Hilton, M.D., and Whiteley, HR. (1982). Nucl. Acids. Res. 10, 2893-
2910.

Daniels, D., Zuber, R., and Losick, R. (1990). Proc. Natl. Acad. Sci. USA 87, 8075-8079.

Dickel, C.D., Burtis, KC, and Doi, RH. (1980). Biochem. Biophys. Res. Commun. 95,
1789-1795.

Dombroski, A.J., Walter, W.A., Record, M.T.Jr., Siegele, D.A., and Gross, CA. (1992).
Cell 70, 501-512.

Flores, 0., Lu, H., Killeen, M.T., Greenblatt, J., Burton, Z.F., and Reinberg, D. (1991).
Proc. Natl. Acad. Sci. USA 88, 9999-10003.

Gaboriaud, C., Bissery, V., Benchetrit, T., and Mornon, J .P. (1987). FEBS Letters 224,
149-155.

Gardella, T. Moyle, H., and Susskind, M.M. (1989). J. Mol. Biol. 206, 579-590.

Garrett, K.P., Serizawa, H., Hanley, J .P., Bradsher, J .N., Tsuboi, A., Arai, N., Yokota, T.,
Arai, K., Conaway, R.C., and Conaway, J .W. (1992). J. Biol. Chem. 267, 23942-23949.

Gong, D.-W., Hashimoto, S., Wada, K., Roeder, R.G., Nakatani, Y., and Horikoshi, M.
(1992). Nucleic Acids Res. 20, 6414.

Gribskov, M. and Burgess, RR. (1986). Nucl. Acids Res. 14, 6745-6761.

Helmann, J .D. (1994). Transcription: Mechanisms and Regulation, edited by Conaway,
R.C., and Conaway, J .W., Raven Press, New York, 1-17.

Helmann, J.D., and Chamberlin, M.J. (1988). Ann. Rev. Biochem. 57, 839-872.
Henrissat, B., Popineau, Y., and Kader, J .C. (1988). Biochem. J. 255, 901-905.

Henry, N.L., Sayre, M.H., and Kornberg, RD. (1992). J. Biol. Chem. 267 , 23388-23392.
Hilton, M.D., and Whiteley, HR. (1985). J. Biol. Chem. 260, 8121-8127.

Hinkle, DC, and Chamberlin, M.J. (1972). J. Mol. Biol. 70, 157-185.

Hyde, E.I., Hilton, M.D., and Whiteley, HR. (1986). J. Biol. Chem. 261, 16565-16570.
Ishihama, A. (1992). Mol. Microbiol. 6, 3283-3288.

Jones, C.H., and Moran, GP. (1992). Proc. Natl. Acad. Sci. USA 89, 1958-1962.

Jones, C.H., Tatti, K.M., and Moran, CR (1992). J. Bacteriol. 174, 6815-6821.

78

Juang, Y.-L., and Helmann, JD. (1994). J. Mol. Biol. 239, 1-14.

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

Killeen, M., Coulombe, B., and Greenblatt, J. (1992). J. Biol. Chem. 267, 9463-9466.
Lampe, M., Binnie, C., Schmidt, R., and Losick, R. (1988). Gene 67, 13-20.

Lemesle-Varloot, L., Henrissat, B., Gaboriaud, C., Bissery, V., Morgat, A., and Mornon,
J.P. (1990). Biochimie 72, 555-574.

Lesley, S.A., and Burgess, RR. (1989). Biochemistry 28, 7728-7734.

Lonetto, M., Gribskov, M., and Gross, CA. (1992). J. Bacteriol. 174, 3843-3849.
McCracken, S., and Greenblatt, J. (1991). Science 253, 900-902.

Pero, J. Nelson, J ., and Tox, TD. (1975). Proc. Natl. Acad. Sci. USA 72, 1589-1593.

Reed, S.J., Ferguson, J., and Jahng, K.-Y. (1988). Cold Spring Harb. Symp. Quant. Biol.
53, 621-627.

Shuler, M.P., Tatti, K.M., Wade, K.H., and Moran, C.P.Jr. (1995). J. Bacteriol. 177,
3687-3694.

Siegele, D.A., Hu, J .C., Walter, W.A., and Gross, C. (1989). J. Mol. Biol. 206, 591-603.
Sopta, M., Burton, Z.F., and Greenblatt, J. (1989). Nature 341, 410-414.

Tan, S., Garrett, K.P., Conaway, R.C., and Conaway, J.W. (1994). Proc. Natl. Acad. Sci.
USA 91. 9808-9812.

Tan, S., Conaway, R.C., and Conaway, J.W. (1995). Proc. Natl. Acad. Sci. USA 92,
6042-6046.

Tjian, R., Losick, R., Pero, J., and Hinnebush, A. (1977). Eur. J. Biochem. 74, 149-154.

Wade, P.A., Werel, W., Fentzke, R.C., Thompson, N.E., Leykam, J .F., Burgess, R.R.,
Jaehning, J .A., and Burton, Z.F. (1995). Mol. Cell. Biol., in press.

Waldburger, C., Gardella, T., Wong, R., and Susskind, M.M. (1990). J. Mol. Biol. 215,
267-276.

Zhou, Y.N., Walter, W.A., and Gross, G.A. (1992). J. Bacteriol. 174, 5005-5012.

Zuber, P., Healy, J., Carter, HL. 111, Cutting, S., Moran, C.P.Jr, and Losick R. (1989). J.
Mol. Biol. 206. 605-614.

CHAPTER III

FUNCTIONAL DOMAINS OF RAP30

79

80

ABSTRACT

A set of deletion mutants of human RAP30, the small subunit of transcription
factor IIF (TFIIF; RAP30/7 4), was constructed to map functional domains. Mutants were
tested for accurate transcriptional activity, RAP74 binding, RNA polymerase II binding
and TFIIB binding. Transcription assays indicate the importance of both N- and C-
terminal sequences for RAP30 function. RAP74 binds to the N-terminal region of
RAP30 between amino acids 1-98. RAP30 N—terminal sequence between 1-50 and central
sequences between 131-159 and 152-176 are important for RNA polymerase II binding.
TFIIB binds to an overlapping region of RAP30, localized to amino acids 1-176 by an
affinity bead procedure. A minimal interaction domain was located between anrino acids

27 - 1 18 by a more sensitive enzyme-linked immunosorbent assay.

81

INTRODUCTION

Transcription by RNA polymerase H requires auxiliary proteins termed general
transcription factors, including TFHA, TFHB, TFHD, TFHB, TFHF, TFIIH, TFIIJ, TFHS
and SHI/elongin, for accurate initiation and efficient elongation. TFIIA, TFIIB, TFHD,
TFHB, TFHF and TFIIH are involved in the early steps of initiation and promoter
clearance, whereas TFHF, TFHS and 8111 facilitate elongation of RNA chains. Most
studies of the functions of general transcription factors have focused on their interactions
with DNA template, transcript, RNAP II, other general transcription factors and
regulatory factors. It has been postulated that the order of assembly of the initiation
complex begins with the binding of TFHD, TFHA and TFIIB to the TATA box and start
site sequences, followed by the assembly of RNAP H bound to TFHF to form a minimal
transcription machinery capable of accurate initiation on supercoiled templates. The
addition of TFIIB, mm and TFHJ yields a complete basal transcription initiation
complex capable of initiation on linear template (reviewed by Zawel and Reinberg, 1993;
Conaway and Conaway, 1993).

In this study, we focus on the RAP30 subunit of TFHF. TFHF was first identified
and purified by affinity chromatography on a column containing covalently immobilized
RNAP H (Sopta et al., 1985). It was named RAP30/’74, for RNA polymerase H-
associating proteins of 30 and 74 kDa apparent molecular mass. Further studies Show that
TFHF is a heteromeric complex of subunits RAP30 and RAP74 that stimulates both
initiation and elongation of transcription (Burton et al., 1988; Flores et al., 1988, 1990,
1991; Conaway and Conaway, 1989; Conaway et al., 1991; Kitajima et al., 1990). The
RAP30 subunit binds directly to RNAP H, reduces the nonspecific binding of RNAP H to
DNA, and helps bring RNAP H into the pre-initiation complex (Flores et al., 1991;
Conaway et al., 1991; Killeen et al., 1992). RAP30 has also been shown to interact with

82

TFHB, and this interaction may be important for pre-initiation complex assembly (Ha et
al., 1993). RAP30 is required for accurate initiation using an extract transcription system
and linear DNA templates (Chang et al., 1993). With a better defined system and highly
supercoiled templates, RNAP H can accurately initiate transcription in the absence of
TFHF (Parvin and Sharp, 1993; Usheva and Shenk, 1994). RAP74 has been found to be
dispensable for initiation under some conditions (Chang et al., 1993; Parvin and Sharp,
1993; Tyree et al., 1993; Usheva and Shenk, 1994), and required for RNAP H to escape
from the promoter (Chang et al., 1993). TFHF also stimulates the elongation rate of
RNAP H (Bengal et al., 1991; Izban and Luse, 1992).

Several research groups used a mutagenic approach to elucidate the functional
domains of RAP30. It has been proposed that RAP30 contains a N-terminal RAP74
binding region (Yonaha et al., 1993; Frank et al., 1995), a central RNAP H binding region
(McCracken and Greenblatt, 1991), and a cryptic C-terminal DNA binding region (Tan et
al., 1994). Here we report the further characterization of RAP74 binding and RNAP H
binding domains of RAP30. The homodimerization and TFIIB binding domains are also
identified.

83

MATERIALS AND METHODS

Wanna

Fragments of a human RAP30 cDNA (Sopta et al., 1989) were subcloned into
pET23d (Novagen) and transformed into E. coli BL21(DE3) for expression (Wang et al.,
1993). To construct RAP30(1-227), an NcoI to BamHI (blunt: end-filled with Klenow
DNA polymerase I and deoxynucleoside triphosphates) fragment was subcloned between
the NcoI and HincH sites of the vector. To construct RAP30(1-176) an NcoI to XhoI
(blunt) fragment was cloned between the Neal and HincH sites of the vector. These
mutants have a DKLAAALEHHHHHH C-terminal extension. To construct RAP30(1-
118), an NcoI to Pqu fragment was subcloned between the NcoI and XhoI (blunt) sites
of the vector. This mutant has an HHHHHH C-terminal extension. To construct
RAP30(1-98) an NcoI to SstI (blunt) fragment was subcloned between the NcoI and
HincH Sites of the vector. This mutant has a DKLAAALEHHHHHH C-terminal
extension. RAP30(1-249), RAP30(27-249), RAP30(51-249), RAP30(110-249),
RAP30(160-249), RAP30(1-152) and RAP30(27-152) were constructed by PCR
amplification using an upstream primer containing an engineered NcoI site and a
downstream primer containing an engineered NotI site. The NcoI to NotI fragments were
subcloned between the same sites of the vector. These mutants have a AALEHHHHHH
C-terminal extension. The internal deletion mutant RAP30(A131-159) was constructed as
follows. The 1-130 fragment was constructed by PCR amplification using an upstream
primer containing an engineered NcoI Site and a downstream primer containing an
engineered Pst I site, and the 160-249 fragment was constructed by PCR amplification
using an upstream primer containing an engineered PstI Site and a downstream primer

containing an engineered NotI site. These two fragments were ligated at the engineered

84

PstI sites and subcloned between Neal and NotI of the vector. The mutant has a
AALEIIl-HHII-III C-terminal extension.

RAP30(1-249), RAP30(1-227), RAP30(1-176), RAP30(1-152), RAP30(1-118),
RAP30(1-98), RAP30(27-249), RAP30(27-152), and RAP30(A131-159) accumulated
within inclusion bodies and were purified by methods previously described for full-
length RAP30 (Wang etal., 1993). RAP30(51-249), RAP30(110-249) and RAP30(160-
249) were in the soluble fraction and were purified by N i2+-affinity chromatography
(Qiagen). RAP30(110-249) was further purified by Mono S (Pharmacia)
chromatography. Protein concentrations were determined using molar extinction
coefficients calculated on the basis of aromatic amino acid composition (Luthy and
Bisenberg, 1990).

A production vector for human TFHB was the kind gift of D. Reinberg and R.
Tjian and this protein was prepared by a protocol supplied by D. Reinberg (Ha et al.,
1991).

1' ..

The transcriptional activity of RAP30 mutants was tested as previously described
(Wang et al., 1993). A TFHF-depleted extract was prepared by immunodepletion using
anti-RAP30 antibodies. This extract was combined with Adenovirus major late promoter
template DNA (pML digested with SmaI, 50 [lg/ml), RAP74, and RAP30 wild type or
mutant, and pre-incubated for 1 hour, in buffer containing 12 mM Hepes pH 7.9, 12%
glycerol, 60 mM KCl, 12 mM MgC12, 0.2 mM EDTA, and 3.2 mM EGTA. 600 11M
ATP, CTP, UTP and 25 11M 0132P-GTP were added to the reaction and transcription was
allowed to proceed for 30 min. Transcripts were isolated and analyzed by
polyacrylamide gel electrophoresis in 50% (w/v) urea and 1x TBE. The accurately
initiated transcript (217 nucleotides) was quantitated using a Molecular Dynamics

phosphorimager.

85

Glfil . 1° [8913.30

Storage Buffer (SB) was used for gel filtration and all binding reactions. SB
contains 20 mM Hepes pH 7.9, 20 % glycerol w/v, 1 mM EDTA, 1 mM EGTA, and
variable KCl concentration (i.e.. SB 0.1 contains 0.1 M KCl). The purified recombinant
RAP30 mutants were denatured in SB 0.5 containing 4 M urea for 1 hour at room
temperature. The samples were dialyzed with SB 0.5 and subsequently applied onto a
Waters Protein Pak 3008W Sizing column (8.0 x 300mm) previously equilibrated with
SB 0.5. The samples were eluted at 0.5 ml/min, and fractions were collected, precipitated

with 125 [lg/ml Na-deoxycholate and 6% w/v trichloro-acetic acid for 1 hour on ice.

Pellets were collected, resuspended in SDS-PAGE sample buffer and electrophoresed.

E . _ . . .

W. 200 pmol RAP30 and RAP74 were incubated
1 hr at room temperature in 0.5 m1 SB 0.1 containing 0.2% BSA. 10 ul Ni2+ resin, pre-
incubated with SB 0.1 containing 0.2% bovine serum albumin, was added and incubated
with tumbling at 4°C for 1 hour. RAP30 mutants bound the N12+ resin through their
histidine tags. Beads were washed three times with 1 ml SB 0.25. Bound proteins were
eluted with 50 pl SDS-PAGE sample buffer. 10 111 of the eluate was electrophoresed by
15% SDS-PAGE and blotted to nitrocellulose. RAP74 was detected in a Western blot
developed with anti-RAP74 antiserum.

W. calf thymus RNAP H was immobilized on
Affi-gel 10 (Bio-Rad) at a density of about 1 mg RNAP H per ml resin. 50 1.11 RNAP H
beads and 300 pmol RAP30 mutant were incubated at 4°C for 1 hour in 0.5 n11 SB 0.1
containing 0.2% BSA. Beads were washed 3 times with 1 ml SB 0.3, and bound proteins
were eluted in 50 111 SB 0.5. 30 111 of this eluate was analyzed on a 15% SDS-PAGE gel

86

developed with silver nitrate. Affi- gel beads without bound protein ligand were used as a
negative control.

W. TFIIB was immobilized on Affi-gel 10 (Bio-
Rad) at a density of about 1 mg TFHB per ml resin. 20 [ll TFHB beads and 300 pmol
RAP30 mutant were incubated at 4°C for 1 hour in 0.5 ml SB 0.1 containing 0.2% BSA.
Beads were washed 3 times with 1 m1 SB 0.25, and bound proteins were eluted in 50 11.1
SB 0.5. 30 u] of this eluate was analyzed on a 15% SDS-PAGE gel developed with silver
nitrate. Affi-gel beads without bound pr0tein ligand were used as a negative control.

ELISA assays to observe RAP30-TFHB interaction were done as described with
minor modifications (Marsalek and Kaguni, 1994). Microtiter wells (Bectron Dickinson;
Pro-Bind) were coated overnight with the protein to be immobilized (5 ug/ml) in 100 111
50 mM sodium borate pH 9.0, at 4°C. Wells were washed three times with 200 111 PBS
containing 0.2% BSA and 0.05% Tween-20 (PBSBT) and blocked with 200 111 PBSBT
for an hour at room temperature. Proteins were added in 50 111 SB 0.1 containing 0.2%
BSA and incubated for 15 min at room temperature. Glutaraldehyde was added to a final
concentration of 1 % for 30 min to cross-link protein-protein interactions. Wells were
washed three times with 200 111 PBSBT. 100 1.11 of rabbit antiserum (1:1000 diluted)
directed against the mobile phase protein was added to each well and incubated two hours
at room temperature. Wells were washed three times with PBSBT, and 100 111 HRP-
conjugated goat anti-rabbit secondary antibody (Bio Rad; 1:3000 diluted) was added and
incubated for one hour at room temperature. Wells were washed 3 times with PBSBT.
Color was developed with 100 111 50 mM sodium citrate (pH 4.0), 0.03% H202 and 0.4
mg/ml o-phenylenediamine. Color development was stopped by addition of 100 111 4 N
H2S 04. The reaction was measured for absorbance at 490 nm using a plate reader (Bio
Tek Instruments; EL310). All determinations were done in duplicate and reported as

average values.

87

RESULTS
Both N- and C-terminal regions of RAP30 are important for transcription activity

A set of RAP30 deletion mutants was constructed with C-terminal histidine tags
to aid in purification and binding reactions (Fig. 1). Each of the RAP30 mutants was
tested for the ability to stimulate accurate runoff transcription from the Adenovirus major
late promoter (Fig. 2). Full-length RAP30(1-249) was much more active in this assay
than any of the deletion mutants. RAP30(1-249) is distinct from normal RAP30 because
it has a C-terminal histidine tag. RAP30(1-227) and (1-176) supported a much lower
level of activity. RAP30(1-152) had barely detectable activity and RAP30(1-118) was
inactive (data not shown). These results are consistent with previously published data
from the Conaway laboratory that indicate that the C-terminus of RAP30 is involved in
DNA binding and is important for accurate initiation (Garrett et al., 1992; Tan et al.,
1994). Activity was also very sensitive to deletion of N-terminal sequences. RAP30(27-
249) supported weak activity (Fig. 2), but RAP30(51-249) was inactive (data not shown).
Using a set of RAP30 mutants with short internal deletions, Conaway and co-workers
have recently shown that amino acid sequences between 16-30 and 136-210 are critical
for initiation of transcription (Tan et al., 1995). Our immunodepleted extract system is
somewhat more tolerant of C-terminal region mutations than this reconstituted system

(see Discussion).

Modification at the C-terminus causes RAP30 to behave as a monomer

Attaching a histidine tag to the C-terminus of RAP30(1-249) caused this protein
to stimulate transcription at lower concentrations than RAP30 with no tag, although
activity saturated at a similar level at higher concentrations (Fig. 3). The C-terminal
histidine tag causes freshly renatured RAP30 to behave as a monomer in gel filtration,

whereas unmodified RAP30 is a dimer (Table 1). As a monomer, RAP30 with the C-

88

Figure l. RAP30 deletion mutants. RAP30(1-249) is the histidine-tagged version of
RAP30. Accurate transcription (tx.) was determined from the Adenovirus major late
promoter (Figure 2). RAP74 binding to RAP30 was determined using a N12+-affinity
bead procedure (Figure 4). RNAP H binding to RAP30 was determined using affinity
beads (Figure 5). TFHB binding to RAP30 was determined by affinity bead and ELISA
procedures (Figures 6 and 7). (+) indicates high activity; (+/-) indicates low activity, (7)
indicates barely detectable activity; and (-) indicates no detectable activity; (n.d.)
indicates that no determination was made for a particular mutant; (*) indicates data is not

shown in this report.

89

33E

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.\+ e+ 6.: 6.: 6.: H _/\: _ mm 75 3
6.: - + - o H . _ ~m TAN
t - 6.: 6.: 6.: r . mewém H
t 1 ~ . t o H 1 J thrO— —
t - ~ - o F . mew- Hm
... - + - 2.0— . _ . _ m¢~-u~
|\+ I I + c . II — mm- F
t\... t l\+ + o F H m — 7p
4+ 4+ -1 + as a H J ...m E
+ + + + XON H 4 ms 7—
6.: 6.: + ...+ 80m .1 L -~-H
+ + + + 88 HA F H mom-—
msoez <2: 86:... ._ .8 2.93 £3
+ 6.: .... + 800 H F H\\\\\\\N. | 0mg
mew o: 5. mm H
.83 (um .
on... .A as. "A v _
0.5-ED 2?; DC; A E O“ .X g F J.
a .8 mg“: was. 1 96:3 8 __ 1.
III.

9652.8

90

Figure 2. Transcriptional activities of RAP30 deletion mutants. The ability of
RAP30 and RAP30 deletion mutants to support accurate transcription initiation was
determined from the Adenovirus major late promoter. An extract derived from human
HeLa cells was depleted of TFHF by immunoprecipitation with anti-RAP30 antibodies.
This extract was supplemented with RAP74 (3.4 pmol) and the indicated amount of
GRAP30 or RAP30 mutant. The accurately initiated transcript was quantitated using a
phosphorimager. Data is shown only for those mutants for which acctu'ate transcription

was high enough to be quantitated.

91

mvmfiw
mu .... _.
NNN. _.
mVN- w

cm

H 95mm

22:3 529:

 

 

 

 

 

 

 

 

 

our

(%) uogldgrosuerl eternoov

92

Figure 3. C-terminal modification affects RAP30 activity. When full length RAP30
with a Six histidine extension [RAP30(1-249)] was compared to unmodified RAP30 in
run-off transcription assay, a reduced amount of RAP30(1-249) was required to saturate

the assay.

93

a; me? Ill
25 33 Incl

ON

 

Hagar

coed. £22m

m... or

 

 

' 38.

 

ii

 

(%) NOIldIHOSNVHl arvunoov

94

623 Emncfim m .3 356.3% mm; 2.82.. Ho 2963 538.9:
Hcmemaam of. .HEE 00m x 0.8 5:28 m:_~_m Smoom xmn. 530.5 9.325
m 8:0 623% 0.6.5 3368 9.... ..otan :ozatumcmb 6v. 2 md :. coverage
6:: m8: 2 6 :. 683206 263 3:32: 025. 223589. notes: 2:.

 

 

 

 

..oEocoE N.N H mm ..m rmm 8m- Coma—(z
.oEoeoE m.mH oH .. 5% a: H- 5:25.
.6589: .4. .N m H ..NN. Hm 6H H- 5:25.
.6889: NKN HN . HN CNN- Sena—S.
.oEocoE Nam mm :m row 86w- 502$.
62.... ...mm mm :92 8:5. 5.
...... ......

 

 

5:26.: .8 .3 85.53%
3:32.. 5:28 .....Eooé :25. ..o 2.2.... .2822. .. oon

95

terminal tag may more easily make appropriate contacts with other transcription factors

and template in assembly of the pre-initiation complex.

RAP74 binds to the N-terminal region of RAP30 between amino acids 1-98

The RAP74 binding site on RAP30 was mapped using an affinity bead procedure
(Fig. 4). RAP74 without a histidine tag was combined with RAP30 or a RAP30 mutant
that contained a C-terminal tag. The mixture was combined with Ni2+-affinity beads.
After washing, bound proteins were eluted with SDS-PAGE sample buffer and analyzed
in a Western blot developed with anti-RAP74 antiserum. Amino acid sequences between
1-98 of RAP30 were sufficient to observe binding between RAP30 and RAP74.
Attempts to produce RAP30 mutants with more extensive C-terminal deletions were not
successful, so no such mutants were tested. Even the shortest N-terminal deletion (27-
249) of RAP30 was unable to bind to RAP74. These results are very Similar to those
previously reported by Yonaha et al. (Y onaha et al., 1993). In this previous report,
interaction between RAP30 and RAP74 was determined using a 2-hybrid reporter system

in contrast to the direct binding assay shown here.

Both N-terminal and central regions of RAP30 are important for polymerase
binding

RAP30 sequences that are important for binding to RNA polymerase H were
analyzed by binding RAP30 mutants to RNA polymerase H immobilized on agarose
beads. The binding reaction was initially done in SB 0.1 and then beads were washed in
SB 0.3 (Figure 5). These experiments indicate that both N-terminal sequences between
amino acids 1-50 and central sequences, including those between amino acids 131-159,
are important for polymerase binding. Using the 0.3 M KCl wash condition, only
RAP30(1-249) and (1-176) bound RNA polymerase H tightly. Much weaker binding was
observed with RAP30(1-152), (1-118), and (1-98). This result indicates that part of the

96

Figure 4. Sequences between amino acids 1-98 of RAP30 are sufficent for RAP74
binding. RAP74 and RAP30 mutant (200 pmol each) were combined with Ni2+-affinity
beads to bind the histidine tag on the RAP30 mutant. After washing with SB 0.25, bound
proteins were eluted with SDS-PAGE sample buffer and analyzed on a Western blot
developed with anti-RAP74 antiserum.

97

83.81;
.m H H- Cor-E

.mm H- Com-.3
.3 H- 58+:
.3 E809:
3.42 H 58...:
88-539:
...-388+:

sew-$81.:
E

taste: 2

 

 

 

 

91011

8

Figure 4

98

Figure 5. RAP30 sequences between 1-50, 131-159 and 152-176 are important for
RNAP 11 binding. 300 pmol RAP30 or mutants was incubated with affinity beads
containing covalently immobilized RNAP H. After washing with SB 0.3, bound protein
was eluted and analyzed on an SDS-PAGE gel developed with Silver nitrate.

 

-_._ .- -.-_.. _._.=-‘-_-

99

RNAP ll affinity beads (0.3 M KC1 wash)

 

.8 78:48
38-8 :8
Sew-o H :8

8.8. H38
.8388

8m- :8
3— H- 50m
ANm H-Hvom

.8 7:8

sem- 50m

 

 

 

 

 

Figure 5

100

RNA polymerase II binding surface is located between amino acids 152-176. Because
Hydrophobic Cluster Analysis (HCA) indicated that RAP30 might have sequence
similarity to bacterial sigma factors within RNA polymerase-binding subregions 2.1
(Chapter 2, Figure 2), a small deletion mutant was constructed within this region of
RAP30. HCA indicated that this region might include amino acids between positions
144-176. One deletion mutant in this region, RAP30(A131-159), was observed to bind
RNA polymerase II weakly, with comparable apparent affinity to RAP30(1-152), (1-
118), and (1-98).

That RAP30(l-98) binds weakly to RNA polymerase H indicates that N-terminal
sequences are important for binding, and analysis of mutants from which these sequences
are deleted reveals their importance. RAP30(27-249) was severely impaired for
polymerase binding and RAP30(51-249) was not observed to bind. RAP30 sequence
between amino acids 1-27, therefore, is important for binding, and sequence between
amino acids 27-50 is essential, despite the presence of the binding region within the
central portion of RAP30. This observation was of great interest to us, because of our
proposed similarity between human RAP30 and homology subregion 1.2 of bacterial
Sigma factors (Chapter 2, Figure 1). This proposed similarity region lies between RAP30
amino acids 39-71, and this is the same region of RAP30 that interacts with RNA
polymerase H. The function of Sigma subregio'n 1.2 is not known, but we predict based
on our results with RAP30 and our HCA analysis, that this region of sigma factors may

contribute to polymerase binding.

Multiple contacts within the RAP74-binding and polymerase-binding regions of
RAP30 contribute to TFIIB binding

RAP30 deletion mutants were tested for binding to TFHB (Fig. 6 and Fig. 7).

Binding was assayed using two methods that may differ in the sensitivity of detection for

101

Figure 6. RAP30 sequences between amino acids 1-176 are required for tight
binding to TFIIB. RAP30(1-152) binds weakly to TFIIB. .300 pmol RAP30 (30) was
incubated with affinity beads containing covalently immobilized TFIIB (HB). After
‘ washing with SB 0.25, bound protein was eluted and analyzed on an SDS-PAGE gel

developed with silver nitrate.

 

102

IIB affinity beads

 

8m- 50m

.8 :8

3m H-596.

3n TCOm

88.8 :8

AmvwroH Son

8%. H88

Hm¢~-n~vom

84.658

 

 

 

 

Figure 6

103

Figure 7. A minimal TFHB-binding region of RAP30 maps between amino acids 27-
118. RAP30 or RAP30 mutants (5 ug/ml) were immobilized in the wells of a microtiter
dish. After blocking, increasing amounts of TFHB were incubated in the wells as
indicated. Binding was detected with anti-TFHB antiserum. The CD. (490 nm) varies
between experiments because of variable time for color development. Wild type is

RAP30 with no histidine tag. RAP30(1-249) has a C-terminal histidine tag.

 

53.353333
Quip—moss:

m¢~roHH Inl-
m rem-H... 111!

l Nm EN Ilal

mVNthN III-1|
mtmtp .Iloll

cube—Hillel

floahh

~—

 

 

 

(Wu 061') '0'0

H 95m...

Ham-.35";
0.3395050:

3H Ilell
up pt— Ilarlt
um 7H loll
2. TH Ilsl
b-tp incl

083; 1101'
309....

NJ

 

 

 

 

 

(W 069) 'CI’O

105

protein-protein interactions. For the experiment shown in Figure 6, TFIIB was covalently
immobilized on agarose beads and used as an affinity adsorbant for RAP30 mutants.
Binding reactions were done at 0.1 M KCl and washed at 0.25 M KCl. RAP30 and
RAP30(1-176) bound most tightly to TFHB (lanes 1 and 6). RAP30(1-152) also bound
but much less efficiently (lane 7). None of the other deletion mutants was observed to
bind. Apparently, an extensive surface of RAP30 contributes to TFHB binding.

In Figure 7, an ELISA plate test was used to measure RAP30-TFIIB binding.
Binding, in this case, was determined in buffer containing 0.1 M KCl. Microtiter plate
wells were coated with RAP30 or a RAP30 mutant. After blocking, TFHB was incubated
with protein bound to the wells. After glutaraldehyde cross-linking and washing, bound
TFHB was detected with anti-TFHB antiserum, using a horseradish peroxidase-linked
second antibody and a colorimetric enzyme assay. Although interpretation of this
experiment requires that binding of mutant RAP30 to microtiter wells be of similar
efficiency (The unshown data suggest that RAP30 mutants appear to bind to microtiter
wells with similar efficiency), ELISA appears to provide a reliable measure of interaction
and a somewhat more sensitive detection of RAP30-TFIIB binding. By this analysis,
RAP30(1-249), (1-227), (1-176), (27-249), and (27-152) bound TFIIB most tightly.
RAP30(1-152) and (1-118) showed reduced affinity. RAP30(51-249) and (110-249)
were further reduced in affinity, and RAP30(l-98) was not observed to bind. Based on
ELISA, therefore, the core of the RAP30-TFIIB interaction may lie between amino acids
27-118 of RAP30, and sequences between 1-176 conuibute to binding. These results

appear to conf'mn and extend the results of the affinity bead binding procedure.

106

DISCUSSION

RAP30 appears to consist of three functional regions (Fig. 8). The N-terminal
region binds RAP74 (Fig. 4) and contributes to RNA polymerase H binding (Fig. 5), the
central region binds RNA polymerase II (Fig. 5, McCracken and Greenblatt, 1991), and
the C-terminal region binds DNA (Tan et al., 1994). In the current work, we have
mapped the functional domains of human RAP30 that are required for accurate
transcription, that bind RAP74, that bind RNAP II and that bind TFIIB (see Figs. 8).

Yonaha et a1. previously reported the RAP74 binding region of RAP30 (Y onaha
et al., 1993). Our determination is consistent with theirs except that we have located a
slightly less extensive surface on RAP30 for binding to RAP74. They mapped the region
of 1-110 of RAP30 as the minimum sequence essential for interacting with RAP74 in
viva using a 2-hybrid gene reporter system. Our determination was based on direct in
vitro contacts between purified proteins and we show that the N-terminal region of
RAP30 between amino acids 1-98 is sufficient for RAP74 binding (Fig. 4). RAP30(27-
249), which does not bind tightly to RAP74 (Fig. 4) or RNAP H (Fig. 5), has weak
residual transcriptional activity (Fig. 2), so contacts with other factors and template may
allow inefficient assembly of an active complex with this mutant. Since our assay is in an
extract system, TFIIF is required both for initiation and elongation of the transcript, so
mutants can potentially be defective in one or both of these processes (Chang et al.,
1993).

TFIIB binds to an extensive region of RAP30 between amino acids 1-176 (Fig. 6).
A much weaker interaction may be maihtained with amino acids between 27-118,
because RAP30(27-249) and (1-118) appear to interact weakly with TFIIB (Fig. 7).
Apparently, multiple contacts within the RAP74-bindin g and RNA polymerase II-binding
regions of RAP30 contribute to TFIIB binding. The central region of RAP30 is weakly

107

Figure 8. Functional domains of RAP30.

108

 

 

 

TF'lelndlna homodimerization
I6 - -
1 98 131 ‘176 249
WNW ‘ pollblndng DNAblndlng
polllblndlng
Ie 4|
mummnactlvlty

FigureS

109

similar to the core binding region of bacterial sigma factors (Chapter II), suggesting that
this region may be involved in RNAP II binding. Consistent with this suggestion, at least
one of the serines residues at positions 135, 136, or 142 is protected from
phosphorylation when RNAP II binds RAP30 (McCracken and Greenblatt, 1991). We
further demonstrated that sequences between 131-159 and 152-176 are important for
RNAP II binding (Fig. 5). This is the first direct evidence that the central region of
RAP30 is indeed involved in RNAP II binding. Interestingly, the N-terminal region of
RAP30 binds to RNAP II weakly, and deletion of the N—terminus abolishes the RNAP II
binding activity (Fig. 5). Since deletion of the N-terminal region also eliminates RAP74
(Fig. 4) and TFIIB (Fig. 6) binding, and activates the DNA binding of the C-terminal
region (Tan et al., 1994), we propose that in addition to binding RAP74 and RNAP II, the
N-terminal region may be a regulatory domain that affects accessibility of central and C-
terminal regions. The sigma subregion 1.2 similarity in this region may be important for
this regulatory function as this region of sigma factors has been proposed to mask DNA
binding by central and C-terminal sigma sequences (Chapter II).

The Conaway laboratory (Tan et al., 1995) has recently published an extensive set
of short internal deletion mutants of RAP30 which they have tested for function in
accurate initiation and elongation. They mapped a region between amino acids 16-30
that was critical for RAP74 binding, accurate initiation, and stimulation of elongation.
Since both RAP30 and RAP74 are required in the reconstituted accurate initiation assay
and in stimulation of the dC-tailed template elongation assay (Tan et al., 1994b), blocking
RAP30-RAP74 interaction can inhibit both initiation and elongation. An adjacent
deletion mutant from amino acids 31-45 fails to stimulate elongation but supports
accurate initiation. Based on our results, we expect that these mutations also affect
interactions with RAP74 and polymerase. Additional transcription factors (ie. TFIIB) in
the reconstituted accurate initiation assay may compensate for suboptimal contacts

between TFIIF subunits or between TFIIF and RNAP II. Since the dC tailed template

110

elongation assay is missing factors other than polymerase, template and TFIIF, this
system may be more sensitive to these N-terminal region mutations. Mutants between
amino acids 91-120 similarly fail to stimulate elongation on the dC-tailed template but
support initiation. Since these mutants are expected to affect RNA polymerase II
binding, this contact appears most important for elongation, as might be expected. Tight
polymerase binding may be dispensable for initiation because of compensation by other
factors in the reconstituted system. Mutations within the central polymerase binding and
the C-terminal DNA binding regions, amino acids 136-240, eliminate accurate initiation
but do not influence elongation. This result is more difficult to reconcile with our results,
since we observe weak accurate transcription with deletion to 1-176 (Fig. 2). Apparently,
the immunodepleted extract system is more tolerant of C-terminal deletions than the
reconstituted system. Additional factors present in the extract but missing in the more
purified system may partially compensate for RAP30-DNA contacts. Taken together,
results from the Conaway laboratory indicate that for the elongation assay, RAP30
contacts with RAP74 and polymerase are most essential. For the initiation assay, RAP30

contacts with RAP74, polymerase and DNA are essential.

111

REFERENCES

Burton, Z.F., Killeen, M., Sopta, M., Ortolan, L.G., and Greenblatt, J. (1988). Mol. Cell.
Biol. 8, 1602-1613.

Chang, C., Kostrub, C. F. and Burton, 2. F. (1993). J. Biol. Chem. 268, 20482-20489.
Conaway, J .W. and Conaway, R. C. (1989) J. Biol. Chem. 264, 2357-2362.

Conaway, R.C., and Conaway, J .W. (1993). Annu. Rev. Biochem. 62, 161-190.

Conaway, R.C., Garrett, K.P., Hanley, J .P., and Conaway, J.W. (1991) Proc. Natl. Acad.
Sci. USA 88, 6205-6209.

Flores, 0., Maldonado, 8., Burton, Z.F., Greenblatt, J ., and Reinberg, D. (1988) J. Biol.
Chem. 263, 10812-10816.

Flores, 0., Ha, I., and Reinberg, D. (1990) J. Biol. Chem. 265, $629-$634.

Flores, 0., Lu, H., Killeen, M., Greenblatt, J ., Burton, Z.F., and Reinberg, D. (1991) Proc.
Natl. Acad. Sci. USA 88, 9999-10003.

Frank, D.J., Tyree, C.M., George, CR, and Kadonaga, J .T. (1995). J. Biol. Chem. 270,
6292-6297.

Garrett, K. P., Serizawa, H., Hanley, J. P., Bradsher, J. N., Tsuboi, A., Arai N., Yokota,
T., Arai, K., Conaway, R. C. and Conaway, J. W. (1992). J. Biol. Chem. 267, 23942-
23949.

Ha, 1., Lane, W.S., and Reinberg, D. (1991) Nature (Lond.) 352, 689-695.

Ha,I., Roberts, S., Maldonado, B., Sun, X., Kim, L. U., Green, M. and Reinberg,D
(1993). Genes Dev. 7, 1021- 1032.

Izban, M.G. and Luse, D. S. (1992) J. Biol. Chem. 267, 13647-13655.
Killeen, M., Coulombe, B., and Greenblatt, J. (1992) J. Biol. Chem. 267, 9463-9466.

Kitajima, S., Tanaka, Y., Kawaguchi, T., N agaoka, T., Weissman, S. and Yasukochi, Y.
(1990) Nucleic Acids Res. 18, 4843-4849.

Marsalek, J ., and Kaguni, J .M. (1994) J. Biol. Chem. 269, 4883-4890

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

Parvin, J. D. & Sharp, P. A. (1993) Cell 73, 533-540.

Sopta, M. Carthew, R.W., and Greenblatt, J. (1985). J. Biol. Chem. 260, 10353-10361.
Sopta, M., Burton, Z. F. and Greenblatt, J. (1989) Nature 341, 410-414.

112

Tan, S., Garrett, K. P., Conaway, R. C. and Conaway, J. W. (1994). Proc. Natl. Acad.
Sci. USA 91, 9808-9812.

Tan, S., Aso, T., Conaway, R.C., and Conaway, J .W. (1994b) J. Biol. Chem. 269, 25684-
25691.

Tan, S., Conaway, R.C., and Conaway, J .W. (1995) Proc. Natl. Acad. Sci. USA 92, 6042-
6046.

Tyree, C.M., George, C.F., Lira-DeVito, L.M., Wampler, S.L., Dahmus, M.E., Zawel, L.,
and Kadonaga, J .T. (1993). Genes Dev. 7, 1254-1265.

Usheva, A., and Shenk, T. (1994). Cell 76, 1115-1121.

Wang, B. Q., Kosu'ub, C. F., Finkelstein, A. and Burton, Z. F. (1993) Protein Expr. Purif.
4, 207-214.

Yonaha, M., Aso, T., Kobayashi, Y., Vasavada, H., Yasukochi, Y., Weissman, S. M. and
Kitajima, S. (1993). Nucleic Acids Res. 21, 273-279.

Zawel, L. and Reinberg, D. (1993). Prog. Nucl. Acids Res. 44, 67-108.

CHAPTER IV

DYNAMIC INTERACTIONS BETWEEN SUBUNIT S OF
TRANSCRIPTION FACTOR TFIIF AND TFIIB

113

114

ABSTRACT

Both RAP30 and RAP74 bind independently to TFIIB. RAP74 antagonizes the
interaction between TFIIB and RAP30, both by binding to RAP30 and by binding to
TFIIB. Analysis of deletion mutants of RAP74 shows that a C-terminal region between
amino acids 358-517 binds directly to TFIIB. This region of RAP74 also binds to RNA
polymerase H. RAP30, therefore, binds TFIIB only in the absence of the RAP74 subunit.
When the TFIIF complex is intact, TFIIF-TFIIB contact appears to be maintained through
the RAP74 subunit. If these binding relationships are maintained within functional
transcription complexes, dynamic interactions between TFIIF subunits and TFIIB may

be a mechanism to separate RAP30 and RAP74 functions during various stages of the

transcription cycle.

115

INTRODUCTION

A minimal pathway for assembly of pre-initiation complexes on RNA polymerase
II-dependent promoters has been defined in vitro (Buratowski et al., 1989; Maldonado et
al., 1990; Zawel and Reinberg, 1993, 1995; Serizawa et al., 1994). On promoters that
include a TATA box, TBP (TATA-binding protein) binds first to this recognition
sequence. TFIIA facilitates this interaction. TFIIB can enter the complex either by
binding to these template-associated factors or by binding to RNA polymerase II. For a
tight association with the complex, polymerase must first associate with TFIIF, so TFIIB
and TFIIF cooperate to bring polymerase into the pre-initiation complex (Conaway et al.,
1991; Flores et al., 1991; Serizawa et al., 1994; Zawel and Reinberg, 1995). TFHB and
TFIIH are additionally required for accurate initiation from linear DNA templates.

TFIIB is a single polypeptide of 33 kDa (Ha et al., 1991; Mailk et al., 1991) that
can roughly be divided by sequence and mutational analysis into N- and C-terminal
regions. At the N-terminus of TFHB is a probable Zn2+-binding sequence that is
important for pre-initiation complex assembly (Buratowski and Zhou, 1993).
Immediately adjacent to the Zn2+-binding motif is a highly conserved sequence that is
important for selection of transcriptional start sites (Pinto et al., 1994). This N-tenninal
region binds the RAP30 subunit of TFIIF and RNA polymerase II (Ha et al., 1993;
Hisatake et al., 1993), and mutations in this region inhibit assembly of u'anscription
intermediates, because they fail to interact with either TFIIF or polymerase (Buratowski
and Zhou, 1993; Ha et al., 1993; Hisatake et al., 1993; Malik et al., 1993). The C-
terminal region of TFIIB contains two imperfect direct repeats. Near the end of the first
repeat is an amphipathic alpha helix with a basic charged face. Sequence surrounding
this structure is important for binding TBP and the acidic activation domain of herpes

simplex virus VP16, although binding sites for these two proteins are separable by

116

mutation (Roberts et al., 1993; Roberts and Green, 1994). In the absence of other factors,
the N- and C-terminal domains of TFIIB are thought to interact, causing masking of
protein binding regions. Binding of the acidic transcriptional activator VP16 to the C-
terminal region appears to open up the TFHB structure to expose the C-terminal region,
which binds TBP, and the N-terminal region, which binds RNA polymerase II and TFIIF
(Roberts and Green, 1994). Thus, TFIIB forms a bridge between TBP and RNA
polymerase II/I‘FIIF, in assembly of the pre-initiation complex, and the assembly steps
involving TFIIB are a target of transcriptional regulators. Recent determinations of an x-
ray crystal structure of TATA-box DNA with TBP and TFIIB (Nikolov et al., 1995) and a
nuclear magnetic resonance structure of TFIIB (Bagby et al., 1995) support this model for
TFIIB function. These structures indicate that the N-terminal region of TFIIB is
presented as a scaffold for subsequent assembly of RNA polymerase II/TFIIF into the
pre-initiation complex (Nikolov et al., 1995; Bagby et al., 1995).

TFIIF is a heteromeric factor of 28 kD (RAP30) and 58 kD (RAP74) subunits
(Flores et al., 1988; Conaway and Conaway, 1989; Kitajima et al., 1990; Flores et al.,
1990), but there is some indication that in certain contexts these subunits may enter
complexes as separate factors (Flores et al., 1991; Killeen et al., 1992; Chang et al., 1993;
see Discussion).

Accurate initiation has been demonstrated from highly supercoiled templates
using a system consisting of RNA polymerase II, TFIIB, and either TBP (Parvin and
Sharp, 1993), or YYl, which is an initiator binding protein (Usheva and Shenk, 1994).
One implication of these observations is that RNA polymerase II and TFIIB might
minimally suffice to select transcriptional start sites. Consistent with this view, swapping
S. pombe for S cerevisiae TFIIB and RNA polymerase II, in a system otherwise
comprised of S. cerevisiae factors, shifts the position of the transcriptional start to that
characteristic of S. pombe (Li et al., 1994). Also, some sua7 mutants in the S. cerevisiae

gene encoding TFIIB are altered for selection of initiation sites (Pinto et al., 1992). 3:408

117

mutants, in the gene encoding the largest subunit of RNA polymerase II, affect
transcriptional starts in a very similar way to these sua7 mutants (Berroteran et al., 1994).
Interestingly, mutations in the S. cerevisiae SSU71/TFGI gene, which encodes the
homologue of the RAP74 subunit of human TFIIF, suppress abnormal start site selection
in a sua7ssu7l double mutant (Sun and Hampsey, 1995). By itself the ssu71 mutant does
not affect transcriptional starts, so TFHF may suppress the sua7 mutant indirectly through
another general factor. The RAP30 subunit of human TFIIF has been shown to interact
physically with the N-terminal region of TFHB (Ha et al., 1993), so the large subunit of
TFIIF interacts genetically, and the small subunit interacts physically, with TFIIB.

The largest subunit of RNA polymerase II has an interesting carboxy-terminal
domain (CID) which consists of 52 repeats of the consensus sequence YSPTSPS (Corder
and Ingles, 1992; Dahmus, 1994). RNA polymerase II enters the pre-initiation complex
most efficiently in the dephosphorylated IIa state. Within the complex the CTD is
multiply phosphorylated on the SP serines, by a subunit of TFIIH and possibly other CI‘D
kinases. Elongating RNA polymerase II molecules are primarily in the highly
phosphorylated 110 state. So the level of phosphorylation of the CTD may regulate
elongation, and a CTD phosphatase may be important for re-cycling dephosphorylated
polymerase Ila to a promoter after termination.

A CI‘D phosphatase has recently been identified that binds to RNA polymerase 11
(Chambers and Dahmus, 1994; Chambers et al., 1995). This phosphatase interacts with a
region of polymerase distinct from the CTD, and will not dephosphorylate the CTD
unless this domain is covalently linked to polymerase. The RAP74 subunit of TFIIF
stimulates CTD phosphatase activity, apparently through its interaction with RNA
polymerase II. Consistent with this view, the C-terminal region of RAP74 that binds
polymerase is essential for stimulation. The C-terminal region of RAP74 is masked for
phosphatase stimulation (Chambers et al., 1995) just as it is for RNA polymerase II

binding (Wang and Burton, 1995) and for TFIIB binding (this paper). TFIIB suppresses

118

stimulation of phosphatase activity by RAP74, and this is further evidence for functional
interaction between TFHB and RAP74 (Chambers et al., 1995).

Based on these observations, there may be significant coupling of TFIIF and
TFIIB function in transcription. TFHF has functions in both initiation and elongation of
RNA chains. TFIIB has known functions in initiation but may have unrecognized
functions in elongation as well.

We have mapped the region of RAP30 that interacts with TFIIB (Chapter III). In
this report, the region of RAP74 that interacts with TFIIB was also mapped. The RAP30
interaction surface for RAP74 overlaps with the RAP30 interaction surface for TFIIB
(Chapter III). RAP74 binding to RAP30 competes with the interaction between RAP30
and TFIIB. Additionally, fragments of RAP74 that interact with TFIIB but do not bind
RAP30 antagonize the interaction between TFIIB and RAP30. Interaction between
TFIIB, RAP30 and RAP74 may allow for distinct pathways of pre-initiation complex
assembly on different promoters and may allow for separate timing of RAP30 and

RAP74 function in initiation and elongation.

119

MATERIALS AND METHODS

W

RAP30 mutants (Chapter III) and RAP74 mutants (Wang and Burton, 1995) were
prepared as described. A production vector for human TFHB was the kind gift of D.
Reinberg and R. Tjian and this protein was prepared by a protocol supplied by D.
Reinberg (Ha et al., 1991).

P. . - . . .

W. Storage Buffer (SB) was used in binding
reactions. SB contains 20 mM Hepes pH 7.9, 20 % glycerol w/v, 1 mM EDTA, 1 mM
EGTA, and variable KCl concentration (i.e.. SB 0.1 contains 0.1 M KCl). TFIIB was
immobilized on Affi-gel 10 (Bio-Rad) at a density of about 1 mg TFIIB per ml resin. 20
pl TFIIB beads and 300 pmol RAP74 mutant were incubated at 4°C for 1 hour in 0.5 ml
SB 0.1 containing 0.2% BSA. Beads were washed with 1 ml SB 0.25, and bound proteins
were eluted in 50 pl SB 0.5. 30 pl of this eluate was analyzed on a 15% SDS-PAGE gel
developed with silver niuate. Affi- gel beads without bound protein ligand were used as a
negative control.

M were done as described with minor modifications (Marsalek and
Kaguni, 1994). Microtiter wells (Bectron Dickinson; Pro-Bind) were coated overnight
with the protein to be immobilized (5 [lg/ml) in 100 pl 50 mM sodium borate pH 9.0, at
4°C. Wells were washed three times with 200 [.11 PBS containing 0.2% BSA and 0.05%
Tween-20 (PBSBT) and blocked with 200 ul PBSBT for an hour at room temperature.
Proteins in the mobile phase were added in 50 111 SB 0.1 containing 0.2% BSA and

incubated for 15 min at room temperature. Glutaraldehyde was added to a final

120

concentration of l % for 30 min to cross-link protein-protein interactions. Wells were
washed three times with 200 Ill PBSBT. 100 111 of rabbit antiserum (1:1000 diluted)
directed against the mobile phase protein was added to each well and incubated two hours
at room temperature. Wells were washed three times with PBSBT, and 100 111 HRP-
conjugated goat anti-rabbit secondary antibody (Bio Rad; 1:3000 diluted) was added and
incubated for one hour at room temperature. Wells were washed 3 times with PBSBT .
Color was developed with 100 pl 50 mM sodium citrate (pH 4.0), 0.03% H202 and 0.4
mg/ml o-phenylenediamine. The development was stopped by addition of 100 111 4 N
H2804. The reaction was measured for absorbance at 490 nm using a plate reader (Bio
Tek Instruments; EL310). All determinations were done in duplicate and reported as
average values.

MBWwas used to detect the interactions between TFIIF
subunits and TFIIB. Histidine-tagged RAP30 or RAP30(1-176) (300 pmol) was
incubated with TFHB (600 pmol) 1 hr at room temperature in 0.5 ml SB 0.1 containing
0.2% BSA, in the presence or absence of RAP74 (600 pmol). 10 Ill Ni2+ resin, pre-
incubated with SB 0.1 containing 0.2% BSA, was added and incubated with tumbling at
4°C for 1 hr. RAP30 bound the Ni2+ resin through their histidine tags. Beads were
washed three times with 1 ml SB 0.25. Bound proteins were eluted with 50 ul SDS-
PAGE sample buffer. 10 ul of the eluate was electrophoresed by 15% SDS-PAGE and
blotted to niu'ocellulose. TFIIB or RAP74 was detected in a Western blot developed with

anti-TFIIB or anti-RAP74 antiserum.

121

RESULTS

Both RAP30 and RAP74 bind independently to TFIIB

A set of RAP30 and RAP74 deletion mutants was constructed with C-terminal
histidine tags to aid in purification and binding reactions (Fig.1; Chapter III; Wang and
Burton, 1995). Because of the genetic and physical interaction between TFIIF and TFIIB
(Ha et al., 1993; Sun and Hampsey, 1995), RAP30 and RAP74 were tested for direct
binding to TFIIB (Fig. 2). TFIIB is shown to bind directly and independently to both
RAP30 (lane 2) and RAP74 (lane 4). This result confirms the previous report of a direct
interaction between TFIIB and RAP30 (Ha et al., 1993) and demonstrates for the first
time that TFIIB also interacts directly with RAP74.

C-terminal region of RAP74 between amino acids 358-517 binds directly to TFIIB
To demonsu'ate the specificity of the TFHB-TFHF interactions and to indicate
their functional importance, the sequences of RAP30 and RAP74 that are important for
binding TFIIB were mapped. RAP30 deletion mutants have been tested for binding to
TFIIB (Chapter III). A set of RAP74 mutants was also tested for binding to immobilized
TFIIB (Fig. 3). RAP74(358-517) bound most tightly (lane 4). Full length RAP74, and
RAP74(87-517) and (Al37-356) bound with somewhat lower affinity (lanes 1, 2 and 6).
RAP74(207-517) and (407-517) bound much more weakly. RAP74 mutants from which
C-terminal sequences were removed bound TFIIB weakly or were not observed to bind
TFIIB (Fig. 4). The TFIIB binding site on RAP74, therefore, appears to be located within
the C-terminal region between amino acids 358-517. .
Sequences within the N-terminal region and central region of RAP74 affect
masking of the C-terminal domain for RNA polymerase II binding (Wang and Burton,
1995) and CTD phosphatase stimulation (Chambers et al., 1995). Since RAP74(358-517)

122

l. RAP30 and RAP74 deletion mutants. RAP30(1-249) is the histidine-tagged version
of RAP30. Accurate transcription (tx.) was determined from the Adenovirus major late
promoter (Figure 2,Chapter 1H). RAP74 binding to RAP30 was determined using a
Ni2+-affinity bead procedure (Figure 4, Chapter III). TFIIB binding to RAP30 (Figures 6
and 7, Chapter III) and RAP74 (Figures 3 and 4) was determined by affinity bead and '
ELISA procedures. (+) indicates high activity; (+/-) indicates low activity; (7) indicates
barely detectable activity; and (-) indicates no detectable activity; (n.d.) indicates that no .

determination was made for a particular mutant; (*) indicates data is not shown in this

report.

123 '

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m tat-tang
if 4: ELISA u. m
:= >: m but!
1 00 1 10 176 249
W30 —I\\\\\\\\I fl 10096
W74 My pd I bhdna DNA Undue
H—->l
ad I ”in
1-249 l 1 >100“
1-227 g I 3096
1476 l fi 20“
1-152 1 fl (< 5")
1-1 10 L I - 0
1-90 I fl 0
27-249 1* I 1096
51-249 1* 1
1 10449 ; J
ISO-249 I fl MI.
27-152 L r o
TF8 am
1 171 205 350 383 444 517
W74
Imam Mutton pol-um mam
1-517 g 1 ...
87-517 1 J 4.
207-517 g l ...].
350-517 I ] “
407-517 I: +/.
A1374“ :2 l I ...
1409 L 1 ' 70
1-356 I fi 70
1496 l— fl 7*
1-205 L J +/-‘
1-172 p ‘I 7*
1-136 :2 7"
1-75 = -*

Figurel

RAP74 WIS
him hitting
EBA bead
+ + n.d.
+ + +
+" n.d.
4' +
+ +/- +/-
+ +/. -
+ - -
- + _
- 7 _
- 7 -
Mi. Mi. -
- + M].
bled: 530
M
+
+
+/-
...
44-
Ml.
+/--
+/-
+/-
+/-
7

‘ 124

Figure 2. Both RAP30 and RAP74 bind independently to TFIIB. 300 pmol RAP30
(30) (lanes 1 and 2) or RAP74 (74) (lanes 3 and 4) was incubated with affinity beads
containing covalently immobilized TFIIB (11B) or no protein ligand (CI‘ for "control").
After washing with SB 0.25, bound protein was eluted and analyzed on an SDS-PAGE

gel developed with silver nitrate.

125

 

4 O

7 3

D. P

M m

_ _
¢n+m= . _ ..
ea+eo .
am+m= ~
om+eo .

r».

 

 

 

i‘

Figure 2

 

126

Figure 3. RAP74 has a masked binding site for TFIIB located within a C-terminal
region between amino acids 358-517. RAP74 and RAP74 mutants were bound to TFIIB
immobilized on agarose beads as described in Figure 2. Bound proteins were analyzed in
a polyacrylamide gel developed with silver nitrate. None of these proteins bound to

negative control beads (data not shown).

 

127

B affinity beads

 

 

Awmmtnm 53K.

C Etnovgn

C. pmtmmmku -

c 3.38:

 

C. pmtnmveR

$5-5:

 

 

Figure 3

128

Figure 4. C-terminal deletion mutants of RAP74 do not bind TFIIB tightly. RAP74

mutants were bound to TFIIB immobilized on agarose beads as described in Figure 2.

 

_.__ ..-...—

129

Ameém Stu

891::

83.5:

AmeAXVN.

$8-5:

ANNTCVN

8m 7::

Amnévwn

 

 

at.

 

 

Figure 4

130

appears to bind more tightly to TFIIB than full-length RAP74, or RAP74(87-517), (207-
517), and (15137-356), the C-terminal domain also appears to be masked for TFIIB
binding. RAP74(207-517) shows the most dramatic masking effect for polymerase
binding (Wang and Burton, 1995) and phosphatase stimulation (Chambers et al., 1995)
just as it does for TFIIB binding (Fig. 3, lane 3).

RAP74 antagonizes the interaction between TFHB and RAP30

Since RAP74 and TFIIB interact with overlapping regions of RAP30 (Chapter
III), RAP74 might stimulate or antagonize TFIIB binding. In Figure 5, an ELISA test for
interactions between TFIIB, RAP30, and RAP74 was done in which one protein was
immobilized in the well of the microtiter plate, another was added to the well as a binding
partner, to be detected with antibody, and the third was added as a potential competitor or
facilitator of the binding interaction. Using this protocol, RAP74 was shown to inhibit
TFHB binding to RAP30 (filled circles). This same conclusion was obtained using a
Ni2+ affinity bead procedure (Fig. 6A). On the other hand, RAP30 does not dissociate
the interaction between RAP74 and TFIIB (x-x). This was perhaps the expected result
because the N-terminal region of RAP74 binds to RAP30 (Y onaha et al., 1993; Wang and
Burton, 1995) while the C-terminal region of RAP74 binds to TFIIB (Fig. 3-4), so both
contacts could be maintained simultaneously. RAP30-RAP74 interactions are also
maintained in the presence of TFIIB as a potential competitor (Fig. 5 (open circles) and
6B). Taken together, these data indicate that interactions between RAP30 and RAP74
take precedence over those between RAP30 and TFIIB. On the other hand, the C-
terminal domain of RAP74 can bind to TFIIB when RAP74 is complexed with RAP30, in
intact TFHF. So if the RAP30 subunit of TFHF were to assemble into a transcription
complex in the absence of RAP74, RAP30 might be expected to interact with TFIIB. If
TFIIF enters as a RAP30/74 complex, however, the C-terminal region of RAP74 is

131

Figure 5. Dynamic interactions between TFIIF subunits and TFIIB. RAP74 blocks
formation of a RAP30-TFIIB complex (filled circles). TFHB does not block formation of
a RAP30/'74 (TFIIF) complex (open circles). RAP30 does not block formation of a
RAP74-TFIIB complex (x-x). TFIIB or RAP74 (5 pig/ml) was immobilized in the wells
of a microtiter plate. A mixture of a mobile protein (3.5 pmol) and increasing quantities
of a potential binding competitor (or facilitator) were added. Detection of bound protein

was with anti-RAP30 or anti-RAP74 antiserum.

 

132

2 $-32 E23838
om :oanEoo

E 229m 2322

m... “222.... 82.532.
III

.2 82:2 ”eezeaeo
m: aegsasoo

om 222a. 2522

E 229a 82.3222.

. loll
n< om-=c< E28630
en :05an00

8 222a 2322
2. 229a 8238.5.

II..I|..

bin

mitt:

8

mafia

22:3 £22m 3:28:50
cm or o

 

 

 

 

 

m 0f.

3“?—

.3.
. ht»

 

(mu 0617) '070

133

Figure 6. A) RAP74 blocks formation of a RAP30-TFIIB complex. A Ni2+-affinity
bead procedure was used, as described in Materials and Methods. Histidine-tagged
RAP30 or RAP30(1-176) (300 pmol) was bound to TFIIB (600 pmol) in the presence or
absence of RAP74 (600 pmol). In the presence of RAP74, TFIIB did not bind to the
Ni2+ resin. A Western blot is shown developed with anti-TFIIB antiserum. B) TFIIB
does not block formation of a RAP30/74 (TFIIF) complex. Histidine-tagged RAP30
or RAP30(1-176) retained RAP74 in both the presence and absence of TFHB. A Western

blot is shown developed with anti-RAP74 antiserum.

134

sick 7 pvom+¢n
8n T 50ml;

3.78th Semi;
Ameut Fvom+v~

2+3“ 7585..
82-582.

vuimwwt pvom+m=
Sew- 58.5..

 

 

 

 

RAP74 ...... .. .... ...

 

 

 

 

Figure 6

135

expected to interact with TFIIB, and interaction between RAP30 and TFIIB is expected
to be blocked.

RAP74 disrupts the interaction between TFIIB and RAP30 by two mechanisms
RAP74 could potentially disrupt the interaction between TFIIB and RAP30 by
two mechanisms, binding to RAP30 and/or binding to TFHB. In Figure 7, both
mechanisms are shown to contribute to blocking the TFIIB-RAP30 interaction. RAP74
mutants containing a RAP30 binding region disrupt TFIIB-RAP30 binding (left panel),
and RAP74 mutants that are missing the RAP30-binding region (amino acids 1-172) but
contain the TFHB-binding region (amino acids 358-517) also disrupt TFIIB-RAP30
binding (right panel). There is a close correlation between the RAP74 mutants that bind
TFIIB most strongly and the ability of these mutants to compete the TFHB-RAP30

interaction (Figs. 3 and 7 (right panel)).

RAP74-TFHB interaction is maintained in complexes containing polymerase

Since the RAP74 interaction appears to take precedence over the RAP30
interaction with TFHB, one question that arises is whether the RAP74-TFIIB interaction
is maintained in complexes containing RNA polymerase II, for instance within a fully
assembled pre-initiation complex. Although this question is difficult to address directly
with a simple experiment, we have tested the effect of RNA polymerase H on binding of
TFHB and RAP74 (Fig. 8). Since both TFIIB and RNA polymerase II bind to
overlapping regions within the C-terminal domain of RAP74 (amino acids 358-517 vs.
363-444), these proteins might compete or cooperate for binding. RNA polymerase II
does not appear to strongly compete or facilitate the TFIIB-RAP74 interaction. When
RAP7 4 was immobilized and TFIIB was added as the binding partner, addition of RNA
polymerase II was not observed to affect formation of complexes containing TFIIB and

RAP74 (filled circles). When TFIIB was immobilized and RAP74 added as the binding

136

Figure 7. RAP74 blocks formation of the RAP30-TFIIB complex both by binding to
RAP30 and by binding to TFIIB. RAP74 or RAP74 mutants were tested in the ELISA
competition assay Figure 5. The region of RAP74 that binds RAP30 maps between
amino acids 1-172 (Wang and Burton, 1995). The TFIIB binding region maps between
358-517 (Fig. 3).

 

 

 

 

 

 

 

 

 

 

20 . . - r .
0 5 10 15
Competltlvertielnmmol)

zo . . . . - ' l ‘
0 5 10 15
CompetitivertelMpmol)

- Figure?

138

Figure 8. RNA polymerase II does not appear to block TFIIB-RAP74 interactions.
This experiment was done essentially as described above for Figure 5 and as indicated in

the figure key.

139

o< 3.2.2 ”c2828
9. 222828

em ”5295 25223

__ d<zm H£9.22 35.3068.
IIIIIII

n< 9725.. E02023

E 2228230

2. 222a 25o:

__ m<zm “5295 35.3065.
lull

2 2.22 ”52828

__ d<zm 222828

E ”520...". 22.02

m: ӣ22m noN___noEE_
...IOIII

2 2.2.2 E2828

__ d<zm 222858

2. 229a 2.82

an ”529d 23:395....
It'll

magma

22:3 £9.05 3228800

o_.

0.0

 

 

/

l
‘0.
O

I
q
,_

 

m...

 

(um car) “do

140

partner, addition of RNA polymerase II caused a moderate reduction in the retention
signal (open circles). Of course, RNA polymerase II is expected to interact with both
TFIIB and RAP74, and polymerase may interfere with binding between anti-RAP74
antibody and RAP74. The slight apparent inhibition of binding that is observed, in the
experiment in which TFIIB was immobilized (open circles), could be attributable to
antibody interference or distribution of TFHB-polymerase and RAP74-polymerase
complexes. It may be difficult to exchange TFHB and/or RAP74 bound to separate
polymerase molecules into a ternary protein complex, since this requires dissociation of
one factor from polymerase. Consistent with this idea, when RNA polymerase II was
immobilized and competition for binding was between RAP74 and TFIIB, a smaller
reduction in the retention signal was observed. This was true whether detection was for
TFIIB (x-x) or RAP74 (filled squares). Of course, when RNA polymerase II is
immobilized, TFIIB and RAP74 could bind independently without maintaining a TFIIB-
RAP74 interaction. Additional experiments will be required to demonstrate TFIIB-

RAP74 contacts within the pro-initiation complex.

'141

DISCUSSION

There is mounting evidence for functional interaction between TFIIF and TFIIB.
These factors cooperate to bring RNA polymerase II into the pre-initiation complex
(Conaway et al., 1991; Zawel and Reinberg, 1993, 1995; Serizawa et al., 1994). TFIIF,
TFHB, and RNA polymerase II cooperate to select transcriptional start sites (Pinto et al.,
1992; Li et al., 1994; Berroteran et al., 1994; Sun and Hampsey, 1995). These factors
may also cooperate to control dephosphorylation of the CTD (Chambers et al., 1995).

Both the RAP30 and RAP74 subunits of TFIIF conuibute to assembly of the pre-
initiation complex. The RAP30 subunit is sufficient to bring RNA polymerase II to the
complex (Flores et al., 1991), but the RAP74 subunit helps to stabilize this interaction
(Tan et al., 1994) and to position RAP30 on template DNA (Coulombe et al., 1994). In
some in vitro transcription systems, both the RAP30 and the RAP74 subunit are required
for initiation of transcription (Tan et al., 1994), but in an extract system from which
TFIIF was removed by immunoprecipitation, only addition of RAP30 was required to
reconstitute initiation (Chang et al., 1993). RAP74 was required in this system, however,
for early elongation of the transcript. It is possible that a trace of RAP74 remaining after
immunodepletion was sufficient to support initiation but not elongation, but no evidence
for differential concentration requirements for RAP74 in initiation and early elongation
has yet been presented, and such a model requires that RAP74 cycle from the complex
after initiation and then reassociate with the complex to support elongation. In any event,
our studies showed that in an extract system RAP74 is required for polymerase to escape
the promoter. Both RAP30 and RAP74 are required for TFIIF to stimulate elongation
strongly using dC-tailed template assays (Tan et al., 1994; Kephart et al., 1994), so we
assume both subunits participate in promoter escape. In one report, weak stimulation of

elongation was seen with RAP74 alone (Kephart et al., 1994). Both RAP30 and RAP74

142

can bind to RNA polymerase II (McCracken and Greenblatt, 1991; Wang and Burton,
1995; Chapter III) and TFIIB (Chapter III; Fig. 2) independently, so mechanisms can be
imagined in which TFIIF subunits might enter transcription complexes separately or, in
some cases, function independently.

RAP30 appears to consist of three functional regions. The N-terminal region
binds RAP74 and RNA polymerase II (Y onaha et al., 1993; Tan et al., 1995; Chapter III),
the central region binds RNA polymerase II (McCracken and Greenblatt, 1991; Chapter
III), and the C-terminal region binds DNA (Tan et al., 1994b). The N-terminus of RAP30
may be involved in regulating the accessibilities of central and C-terminal regions
(Chapter III). RAP74 appears to be divided into three functional domains, as indicated by
sequence analysis (Finkelstein et al., 1992; A80 et al., 1992) and deletion mutants (Wang
and Burton, 1995). The RAP74 primary sequence can be divided into an N-terminal
basic region, a highlycharged cenu'al region with overall negative charge, and a C-
terminal basic region. The N-terminal region binds to RAP30 (Yonahaet al., 1993;
Wang and Burton, 1995). The cenu'al region appears to be a largely unstructured hinge
that conu'ols accessibility of the C-terminal region. The C-terminal region binds directly
to RNA polymerase II (Wang and Burton, 1995) and is important for stimulation of a
CTD phosphatase (Chamber et al., 1995). Deletion from the N-terminus of RAP74
initially increases masking of the polymerase binding activity (Wang and Burton, 1995)
and eliminates stimulation of CPD phosphatase activity (Chambers et al., 1995). Further
deletion within the central region unmasks both polymerase binding and CTD
phosphatase activities. The central region is also the site of phosphorylation by casein
kinase II, and phosphorylation by this and/or other kinases appears to stimulate
polymerase binding by TFIIF (Kitajima et al., 1994).

Multiple contacts within the RAP74-binding and RNA polymerase II-binding
regions of RAP30 contribute to TFIIB binding (Chapter III). Addition of RAP74 blocks
interaction between TFIIB and RAP30 through two mechanisms. By binding the N-

143

terminal region of RAP30, the N-terminal region of RAP74 can block interaction
between RAP30 and TFIIB. Also, by binding TFIIB, the C-terminal region of RAP74
can block this interaction. Most likely, the C-terminal region of RAP74 interacts with the
N-terminal region of TFHB to compete with RAP30 binding. The structure of the TATA
box/TBP/I'FIIB complex strongly indicates that interaction between TFIIF and TFIIB
might be through the N-terminal region of TFIIB (Nikolov et al., 1995). Since RAP74
dissociates RAP30-TFHB interactions, TFIIF/TFIIB contact is most likely maintained
through the C-terminal region of RAP74.

The strongest contacts between TFIIB and RAP74 are within the C-terminal
region of RAP74 between amino acids 358-517 (Fig. 3). The C-terminal region of
RAP74 shows a very similar pattern of masking by N-terminal and central region
sequences for TFIIB binding (Fig. 3), RNA polymerase II binding (Wang and Burton,
1995), and CTD phosphatase stimulation (Chambers et al., 1995). RAP74 can bind to
RAP30 and TFIIB simultaneously (Fig. 5). This result is consistent with the mapping of
RAP74 functional domains, since the N-terminal region binds RAP30 and the C-terminal
region binds TFIIB. Although RNA polymerase II and TFIIB bind to overlapping
regions within the C-terminal domain of RAP74, RNA polymerase II does not appear to
strongly compete for RAP74-TFIIB binding (Fig. 8). That TFIIB and RAP74 might
interact in complexes containing RNA polymerase II is also indicated by the genetic
interaction between yeast SUA7 and SSU71 (Sun and Hampsey, 1995) and by TFIIB-
mediated suppression of RAP74 stimulation of CPD phosphatase activity (Chamebers et
al., 1995). Interaction between the C-terminal region of human RAP74 and TFIIB,
therefore, may be maintained in complexes with RNA polymerase II.

The SUA7 gene in yeast encodes TFIIB, and the RAP74 homologue in yeast is
encoded by SSU71/TF6]. sua7 mutants that confer cold sensitivity and altered
transcriptional start site selection, and are suppressed by ssu71, map to a particular region

of TFIIB near the N-terminus (Pinto et al., 1994). From our work, we would have

144

predicted that the ssu71 mutations that suppress sua7 might map within the C-terminal
. region of SS U71 where human TFHB and RAP74 interact; however, the ssu71 G363D
and G363R alleles (M. Hampsey, personal communication) correspond to the K111
position of human RAP74 (Wang and Burton, 1995), within the RAP30 binding region.
So genetics implicate both TFIIF subunits in start site selection. In S. cerevisiae,
transcriptional start sites can be more than 100 bases downstream from a TATA box.
RNA polymerase II appears to "melt in" near the TATA box and scan single-stranded
DNA for the +1 position (Giardina and Lis, 1993). Since TFIIB and TFIIF are part of the
start site selection mechanism, both factors may u'averse a significant length of DNA
from the TATA box to the initiation site.

Although the experiments shown here demonstrate specific protein-protein
contacts between general transcription factors TFHB and TFIIF, the importance of
RAP74 preventing interaction between RAP30 and TFIIB is not yet clear. Based on our
data, TFIIF is expected to interact with TFIIB through the RAP74 subunit, but in the
absence of RAP74, a RAP30-TFIIB complex could be maintained. Such alternate
structures are most easily understood if TFIIF does not invariably function as an intact
unit, but rather, under some circumstances, as two independent factors, RAP30 and
RAP74. In Figure 9, models are presented to describe alternate pathways for initiation
and productive elongation based on dynamic interaction between TFIIB and TFIIF
subunits. These models account for the possibility that RAP30 and RAP74 may have
partially separable functions in initiation and early elongation (Flores et al., 1991; Killeen
et al., 1992; Chang et al., 1993). Also a model is presented that may explain how
dynamic interaction between TFIIB and TFIIF subunits might influence elongation.

According to one scheme (Fig. 9A), TFIIF enters the pre-initiation complex as a
unit and interacts with TFIIB through the C-terminal domain of RAP74. This mechanism
has been extensively demonstrated using in vitro systems, although alternate pathways to

complex assembly are also possible. In this case, RAP74 is present during pre-initiation

145

Figure 9. models for dynamic TFIIF-TFIIB interactions during initiation, promoter
escape, and elongation. A) The most familiar assembly and initiation mechanism. In
this scheme, TFHF enters the pre-initiation complex as an intact factor. Initiation and
promoter escape proceed. Contact between TFIIF and TFIIB, in this case, is through the
RAP74 subunit. B) on some promoters transcription may be regulated at the level of
promoter escape rather than initiation. Initiation can proceed in the absence of RAP74
(Chang et al., 1993). In this case, RAP30 binds TFIIB. Since RAP74 prevents release of
newly-initiated transcripts (C.-h. Chang and Z.F. Burton, submitted), regulatory factors
must stabilize the initiated complex. Promoter escape requires the RAP74 subunit
(Chang et al., 1993), so by timing the entry of RAP74 into the complex, productive
initiation is regulated at the level of promoter escape. C) If TFIIB has a role in
elongation, dynamic interactions between TFHB and TFIIF subunits may be important to
regulate this stage of the transcription cycle. Since intact TFIIF stimulates elongation
rates (Price et al., 1989; Bengal et al., 1991; Izban and Luse, 1992), regulating RAP30-

RAP74 subunit contacts will stimulate or inhibit elongation.

146

 

 

323%

 

a 0.5mm—

ong 28°59..— .n

in!

o it...

man—... onus—

Elam—2H
finds-

 

..euaaa .<

147

complex formation to stabilize the interaction between RAP30 and template DNA (Tan et
al., 1994; Coulombe et al., 1994). This function of RAP74 can be complemented by a
factor or factors present in extract u’anscription systems but missing from highly purified
systems (Chang et al., 1993). In this scheme, RAP74 is present within the newly-initiated
complex to protect the short transcript from release and to drive RNA polymerase 11 into
a productive elongation mode (C.-h. Chang and Z.F. Burton, submitted). Therefore,
transcription is regulated at the level of initiation.

Transcription from some promoters, however, may be controlled by regulating
promoter escape (Fig. 9B). RNA polymerase 11 cannot exit the promoter unless RAP74
enters the complex (Chang et al., 1993). The function of RAP74 in pre-initiation
complex assembly that has been demonstrated in several defined in viuo systems (Tan et
al., 1994; Coulombe et al., 1994) can be replaced by factors present in cell exu'acts
(Chang et al., 1993). In the absence of RAP74, TFIIB binds the N-terminal region of
RAP30. Accurate transcription initiation from linear templates has been demonstrated in
the absence of added RAP74 in extract systems but not in highly purified systems. The
newly-initiated transcript, in this case, requires stabilization. RAP74 can provide this
stabilization (C.-h. Chang and Z.F. Burton, submitted) as can other factors present in
extract systems (Chang et al., 1993). Productive transcription from such a promoter can
be regulated by controlling the stability of the newly-initiated complex and by timing the
entry of RAP74 into the complex to allow promoter escape by RNA polymerase 11.
Other protein factors can affect these mechanisms. For instance, serum response factor
binds to the serum response element upsu'eam of the human c-fos promoter. This factor
regulates transcription through interactions with RAP74 (Zhu et al., 1994; Joliot et al.,
1995), and may therefore function by stabilizing the newly-initiated complex and
facilitating promoter escape.

TFIIB is known to be an initiation factor, but may have unrecognized functions in

elongation as well. Our laboratory became interested in this issue when we isolated RNA

148

polymerase II-binding transcription factors from yeast by anti-CTD affinity
chromatography (Wade et al., 1995). Surprisingly, TFHB and TFIIF were of similar
abundance in complexes with RNA polymerase II. Since anti-CTD chromatography
should yield a mixture of initiation and elongation forms, we expected a much lower
yield of TFIIB relative to TFIIF, if TFHB were only involved in initiation. As discussed
above, the observation of Chambers et al. that TFHB suppresses RAP74-mediated
stimulation of a CTD-phosphatase also links TFHB function either to transcription
elongation or to RNA polymerase II recycling after termination (Chambers et al., 1995).
Zawel et al. have demonstrated that in a purified transcription system, TFIID
remains bound at the promoter, and TFIIB, TFIIF, TFIIE, and TFIIH dissociate after
initiation (Zawel et al., 1995). TFHF then re-associates with elongating polymerase but
TFIIB reassociates with TBP at the promoter. These experiments, however, were done in
a system that is missing many factors that might contribute to the stability of elongation
complexes. In cells and extract systems, therefore, TFIIB and TFIIF may not dissociate
and may remain bound to polymerase during elongation. If this is the case, dynamic
TFHB-TFHF interactions may modulate elongation rates, contribute to pausing, and/or

conu'ol termination and polymerase re-cycling (Fig. 9C).

Acknowledgements

We thank M. Hampsey for helpful comments on this manuscript and for
permission to mention ssu7l G363D and GB63R alleles. We thank D. Reinberg and R.
Tjian for a TFIIB production clone and for instructions for TFIIB purification.

This work was initially supported by a grant from the National Institutes of
Health. This work was also supported by the Michigan State University Agricultural
Experiment Station, by Michigan State University, and by Ms. Verna C. Finkelstein.

149

REFERENCES

Aso, T., Vasavada, H. A., Kawaguchi, T., Germino, F. J., Gangelly, S., Kitajima, S.,
Weissman, S. M. and Yasukochi, Y. (1992). Nature 355, 461-464.

Bagby, S., Kim, S., Maldonado, B., Tong, K.I., Reinberg, D., and Ikura, M. (1995) Cell
82. 857-867.

Bengal, B., Flores, O., Krauskopf, A., Reinberg, D. and Aloni, Y. (1991) Mol. Cell. Biol.
11, 1195-1206.

Berroteran, R.W., Ware, D.B., and Hampsey, M. (1994) Molec. and Cell. Biol. 14, 226-
237

Buratowski, S., and Zlou, H. (1993) Proc. Natl. Acad. Sci. USA 90, 5633-5637.
Buratowski, S., Hahn, S., Guarente, L., and Sharp, RA. (1989) Cell 56, 549-561.
Chambers, R.S., and Dahmus, ME. (1994) J. Biol. Chem. 269, 26243-26248.

Chambers, R. S., Wang, B. Q., Burton, Z. F. and Dahmus, M. E. (1995) J .Biol.Chem.
270, 14962-14969.

Chang, C., Kostrub, C. F. and Burton, Z. F. (1993). J. Biol. Chem. 268, 20482-20489.
Coulombe, B., Li, J ., and Greenblatt, J. (1994) J. Biol. Chem. 269, 19962-19967.
Conaway, J .W. and Conaway, R. C. (1989) J. Biol. Chem. 264, 2357-2362.

Conaway, R.C., Garrett, K.P., Hanley, J .P., and Conaway, J.W. (1991) Proc. Natl. Acad.
Sci. USA 88, 6205-6209.

Corden, J .L., and Ingles, CJ. (1992) in Transcriptional Regulation, McKnight, S.L., and
Yamamoto, K.R., eds., Cold Spring Harbor Laboratory Press, N.Y., pp. 81-107.

Dahmus, ME. (1994) in Transcription: Mechanism and Regulation, Conaway, R.C., and
Conaway, J .W., eds., Raven Press, N.Y., pp. 243-262.

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

Flores, 0., Maldonado, 13., Burton, Z.F., Greenblatt, J ., and Reinberg, D. (1988) J. Biol.
Chem. 263, 10812-10816.

Flores, 0., Ha, I., and Reinberg, D. (1990) J. Biol. Chem. 265, $629-$634.

Flores, 0., Lu, H., Killeen, M., Greenblatt, J ., Burton, Z.F., and Reinberg, D. (1991) Proc.
Natl. Acad. Sci. USA 88, 9999-10003.

Garrett, K. P., Serizawa, H., Hanley, J. P., Bradsher, J. N., Tsuboi, A., Arai N., Yokota,
T., Arai, K., Conaway, R. C. and Conaway, J. W. (1992). J. Biol. Chem. 267 , 23942-
23949.

150

Giardina, C., and Lis, J .T. (1993) Science 261, 759-762.
Ha, 1., Lane, W.S., and Reinberg, D. (1991) Nature (Lond.) 352, 689-695.

Ha, I., Roberts, 8., Maldonado, E., Sun, X., Kim, L. U., Green, M. and Reinberg, D.
(1993). Genes Dev. 7, 1021-1032. '

Hisatake, K., Roeder, R.G., and Horikoshi, M. (1993) Nature (Lond.) 363, 744-747.
Izban, M.G. and Luse, D. S. (1992) J. Biol. Chem. 267, 13647-13655.
Joliot, V., Demma, M. and Preywes, R. (1995) Nature 373, 632-635.

Kephart, D.D., Wang, B.Q., Burton, Z.F., and Price, DH. (1994) J. Biol. Chem. 269,
13536-13543.

Killeen, M., Coulombe, B., and Greenblatt, J. (1992) J. Biol. Chem. 267, 9463-9466.

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

Kitajima, S., Chibazakura, T., Yonaha, M., and Yasukochi, Y. (1994) J. Biol. Chem. 269,
29970-29977.

Li, Y., Flanagan, P.M., Tschochner, H., and Kornberg, RD. (1994) Science 263, 805-
807.

Luthy, R., and Eisenberg, D. (1990) in Sequence Analysis Primer, Gribskov, M., and
Devereux, J ., eds., Stockton Press, N.Y., pp. 61-87.

Maldonado, B., Ha, I., Cortes, P., Weis, L., and Reinberg, D. (1990) Mol. Cell. Biol. 10,
6335-6347.

Malik, S., Hisatake, K., Sumimoto, H., Horikoshi, M., and Roeder, R.G. (1991) Proc.
Natl. Acad. Sci. USA 88, 9553-9557.

Malik, S., Lee, D.K., and Roeder, R.G. (1993) Mol. Cell. Biol. 13, 6253-6259.
Marsalek, J., and Kaguni, J .M. (1994) J. Biol. Chem. 269, 4883-4890
McCracken, S. and Greenblatt, J. (1991) Science 253, 900-902.

Nikolov, D.B., Chen, H., Halay, B.D., Usheva, A.A., Hisatake, K., Lee, D.K., Roeder,
R.G., and Burley, S.K. (1995) Nature (Lond.) 377, 119-128

Parvin, J. D. & Sharp, P. A. (1993) Cell 73, 533-540.
Pinto, I., Ware, D. B., and Hampsey, M. (1992) Cell 68, 977-988.

ginto, 1., Wu, W.-H., Na, J.G., and Hampsey, M. (1994) J. Biol. Chem. 269, 30569-
0573.

Price, D.H., Sluder, A.E., and Greenleaf, A.L. (1989) Mol. Cell. Biol. 9, 1465-1475.

151

Roberts, S.G.E., and Green, MR. (1994) Nature (Lond.) 371, 717-720.

Roberts, S.G.E., Ha, I., Maldonado, B., Reinberg, D., and Green, MR. (1993) Nature
(Lond.) 363, 741-744.

Serizawa, H., Conaway, J .W., and Conaway, R.C. (1994) Transcription: Mechanisms and
Regulation, Conaway, R.C., and Conaway, J .W. eds., Raven Press, N.Y., pp. 27-43.

Sopta, M, Burton, Z. F. and Greenblatt, J. (1989) Nature 341, 410-414.
Sun, Z. and Hampsey, M. (1995) Proc. Natl. Acad. Sci. USA 92, 3127-3131.

Tan, 8., Aso, T., Conaway, R.C., and Conaway, J .W. (1994) J. Biol. Chem. 269, 25684-
25691.

Tan, 8., Garrett, K. P., Conaway, R. C. and Conaway, J. W. (1994b). Proc. Natl. Acad.
Sci. USA 91, 9808-9812.

Tan, 8., Conaway, R.C., and Conaway, J .W. (1995) Proc. Natl. Acad. Sci. USA 92, 6042-
6046.

Usheva, A. & Shenk, T. (1994) Cell 76, 1115-1121.

Wade, P.A., Werel, W., Chang, C.-h., Shi, X., Wolf, A.J., Sherman, J., Fentzke, R.C.,
Thompson, N.E., Leykam, J.F., Reed, S.I., Burgess, R.R., Jaehning, J .A., and Burton,
Z.F. (1995) Molec. Cell. Biol., in press.

Wang, B.Q. and Burton, Z.F. (1995) J. Biol. Chem. 270, 27035-27044.

Wang, B. Q., Kostrub, C. F., Finkelstein, A. and Burton, Z. F. (1993) Protein Expr. Purif.
4, 207-214.

Yonaha, M., Aso, T., Kobayashi, Y., Vasavada, H., Yasukochi, Y., Weissman, S. M. and
Kitajima, S. (1993). Nucleic Acids Res. 21, 273-279.

Zawel, L. and Reinberg, D. (1993) Prog. Nucl. Acids Res. 44, 67- 108.

Zawel, L. and Reinberg, D. (1995) Annu. Rev. Biochem. 64, 533-561.

Zawel, L., Kumar, K.P., and Reinberg, D. (1995) Genes Develop. 9, 1479-1490.
Zhu, H., Joliot, V. and Prywes, R. (1994) J. Biol. Chem. 269, 3489-3497.

MICHIGAN

llflflflllWllfillllllll

U

11111111“

 

29301