ACTIVATION OF RNA POLYMERASE II

MEDIATED TRANSCRIPTION

Andrew Emili

A thesis submitted in conformity with the requirements for

the Degree of Doctor of Philosophy

at the Graduate Department of Molecular and Medical Genetics in the University of Toronto

G Copyright by Andrew Emili 1997

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1 dedicate this work to my wife and my family

Activation of RNA Polymerase II Mediated Transcription by Andrew Emili

A Thesis submitted towards the Degree of Doctor of Philosophy, 1997

Graduate Department of Molecular and Medical Genetics, Universitv of Toronto.

Abstract

I have developed a sensitive and highly selective in oitro crosslinking strategy

to characterize the protein-protein interactions mediated by a sequence-specific

activator of transcription with components of the RNA polymerase II transcriptional

machinery. The basis ot this approach involved the selective modification of the

chirneric transactivator LexA-E2F-1 with the photoreactive crosslinking reagent

maleimide-4-benzophenone at a single cysteine residue located within its activation

domain. Using this approach, I have demonstrated that LexA-E2F-1 can interact in a

direct and binding-site-dependent manner with the TATA-binding protein TBP.

I provided evidence that this interaction is biologically relevant bv showing that

mutations within the E2F-1 activation domain which impair activation by

LexA-E2F-1 also reduce crosslinking of LexA-E2F-1 to TBP.

1 have refined my original crosslinking rnethodology in order to identify

addi tional protein targets of Led-E2F-1 in an in irifro transcription svstem derived

from a veast ce11 extract. Using this approach, 1 have shown that the activation

domain of LexA-E2F-1 interacts in a promoter-dependent manner with a novel

component of the yeas t RNA polymerase II transcrip tional machinerv, XTC1.

The XTCI gene product also interacts directly with the activation domains of the

herpes virion protein VP16 and the yeast activator GAL4, suggesting it is a common

target of activators. Yeast strains deleted for the XTCl gene exhibit growth defects and

altered responses of the RNA polymerase U transcriptional machinery to activators

in vioo consistent with XTCl being a physiologically relevant target of activators in

yeast.

..*

I l l .

Finally, I have performed affinity chromatography experiments aimed at

identifying human proteins which interact with the evolutionarilv conserved

carboxv-terminal domain (CTD) of the largest polypeptide subunit of the RNA

polymerase II. 1 have purified and identified two such CTD-binding proteins as the

essential splicing factor PSF and the putative splicing factor p54nrb. Since splicing of

rnessenger RNA is intimately coupled to the process of transcriptional elongation

in viuo, this observation suggests that the CTD may be directly involved in the

processing of nascent RNA transcripts in addition to its role in regulating

transcription by Pol II.

My graduate experience has been an incredible joumey, one which

made me a stronger, wiser, and more mature person in many wayç. 1 consider

myself very fortunate to have felt, first hand, the tremendous excitement

which comes from discovery and the great persona1 satisfaction which comes

from being creative. I also feel I have leamed how to face any challenge with

faith, hard work, and persistence. Most of all, 1 have loved the opportunity to

nourish the deep fascination 1 have for life. Now, i t brings me great pleasure

to thank the many people who have helped and guided me along this path.

First, 1 would like to express rny gratitude to my supervisor, Jim ingles,

for his tremendous encouragement, support, and patience. I am grateful to

Jim for ailowing me the opportunity to develop as an independent thinker

and scientist. It may not have been easy, but it was this freedom which 1

wanted most from grad school.

Second, 1 wish to express my great affection and gratitude to my

partners in science: Raj Gupta, Mike (Mickey) Shales, Lina Demirjian,

Johnson Wong, Craig Dorrell, Dan Fitzpatrick, Rahim Lapak and (Big) Mike

Kobor. 1 have been enriched by their friendship and will cherish mv

experiences with them.

Third, 1 wish to thank Brenda Andrews, Mike Tyers, and Jack

Greenblatt for their guidance and encouragement throughout my studies - 1

consider them my role models and hope that 1 can live up to their

expecta tions.

Finally, 1 wish to thank my wife, Alia, and Our family for the warmth

and love they have brought into my life - I have only succeeded because of

them.

TABLE OF CONTENTS

CHAPTER I : Introduction.

r. 11.

DI.

IV.

v.

VI.

VII.

VIII.

IX.

Preface

Promoters of Transcription

Sequence-Çpecific Activa tors of Transcription

The Pol II Transcription Cycle .

The CTD .

The Pol II Transcriptional Machinery .

i. The General Transcriptional Factors .

a. Initiation Factor TFIID .

b. The TATA-binding Protein .

c. The TBP-Associated Factors .

d. TFIIB

e. M E , TFIIF, and TFIIH

ii. Transcriptional Cofactors .

a.TheMediator .

b. The SWI/SNF Complex

c. Adaptors .

d. TFIIA

e. Elongation Factors

Models of Transcriptional Activation .

Closing Comments

Thesis Rationale .

References .

vi .

CHAPTER II : Promoter-Dependent Photo-Crosslinking of the Acidic

Transcriptional Activator E2F-1 to the TATA-Binding Protein.

Summary .

Introduction

Experimental procedures .

Results

Discussion .

References .

CHAPTER III : Identification of a Novel Target of Transcriptional

Activators by Pho to-Crosslinking.

Summary .

Introduction

Methods .

Results & Discussion

References .

CHAMER IV : Interaction of the C-Terminal Domain of the Largest Subunit of

RNA Polymerase II with the Essential Splicing Factor PSF and the

Putative Splicing Factor p54nrb.

Surnmary .

Introduction

vii.

Methods -

Resul ts

Discussion .

References .

APPENDIX : The RNA Polymerase II C-Terminal Domain: Links to a Bigger and

Better 'Holoenzyrne'?.

Summary .

Introduction and Discussion .

References .

vii i .

TABLE OF CONTENTS

Figures and Tables

CHAPTER 1 : Introduction.

Fig. 1. Two views of the steps involved in the activation of

transcription at a Pol promoter. 1-34

CHAPTER II : Promoter-Dependent Photo-Crosslinking of the Acidic

Transcriptional Activator E2F-1 to the TATA-Binding Protein.

Fig. 1. Purified transcription factors .

Fig. 2. Promoter-dependent crosslinking of an activator to TBP .

Fig . 3. Si te-specificity of the photo-crosslinking

Fig. 4. Interaction of the activator with TBP at other promoters .

Fig. 5. The degree of crossiinking correlates with transcriptional activation

Fig. 6. Effects of TFIW and TFIIB on the activator-TBP interaction

Fig. 7. Crosslinking of the activator with TFnB

CHAPTER III : Identification of a Novel Target of Transcriptional Activators

by Pho to-Crosslinking

Fig. 1. Selective crosslinking of an activator to proteins in a yeast extract 111-8

Fig. 2. Purification and cloning of XTCl 11140

Fig. 3. XTCl interacts with the activation domains of several activators . 111-12

Fig. 4. XTCl copurifies with the Pol II holoenzyme and is required for

normal ceil growth ID-14

Table 1. Hyperactivation of transcription in XTCI deficient yeast . III-16

CHAPTER IV : Interaction of the C-Terminal Domain of the Largest Subunit of

RNA Polymerase II with the Essential Splicing Factor PSF and the

Putative Splicing Factor p54nrb.

Fig. 1. Expression of the CTD of mouse in recombinant form , IV-8

Fig. 2. Affinity purification of CTD-binding proteins from a HeLa ce11 extract I V 4 0

Fig. 3. CTD kinase activity in the eluate from a CTD affinity column . IV-11

Fig. 4. Purification and identification of two CTD-interacting proteins . IV- 13

Fig. 5 . Binding of PSF and p54nrb to a CTD affinity column . IV-16

CHAPTER I.

INTRODUCTION

I I - 1

PREFACE

Eukaryotes employ three distinct RNA polymerases to catalyze

transcription of nuclear genes. RNA polymerase II (Pol II), which is responsible

for the synthesis of messenger RNA, is by far the most highly regulated of these

enzymes. The activity of Pol II is regulated in a gene-specific manner through the

action of an extensive network of sequence-specific DNA-binding transcription

factors. As this class of regulators plays a particularly crucial role in normal ce11

growth and development, there has been a tremendous effort in recent years

aimed at elucidating the fundamental mechanisms by which they hnction.

In the introductom chapter of my Thesis, I discuss the principle

mechanisms by which a subset of gene-specific transcription factors, known as

transactivators, are thought to stimulate the activity of Pol II at a promoter.

In particular, 1 focus on the experimental evidence supporting a role for multiple

distinct components of the Pol 11 associated hanscriptional machinerv as the

hnctional targets of transactivators. Ln Chapters II & III, I report the results of

experiments 1 have performed to elucidate the specific protein-protein

interactions which occur between transactivators and componentç of the Pol II

transcription machinery. 1 also discuss the implications of my studies for our

understanding of the physiological control of Pol II mediated transcription.

Finally, in Chapter IV, I present the results of experirnents 1 have performed to

identify human proteins which interact with the unique, evolutionarily

conserved carboxy-terminal domain of the largest subunit of Pol II.

1. Promoters of Transcription

A major landmark in the study of the regulation of RNA polymerase II (Pol II)

was the discovery of specific DNA sequences, termed promoters, located at the 5'

ends of al1 mammalian, viral, and yeast protein-coding genes. These promoter

sequences are absolutely required for the enzyme to initiate transcription and

determine a specific start site of transcription (for a review, see Breathnach and

Chambon 1951 and Struhl 1989). Comparative analysis of many promoters has

indicated that most consist of one or more readily identifiable conserved sequence

elements which mediate the ability of Pol 11 to initiate transcription i i l cioo and irz

i>itt-o. These elements include an adenine -thymidine-rich sequence known as the

TATA box (concençuç TATa / Wa/ t; Breathnach and Chambon 1981) and /or a

pyrimidine-rich sequence known as the initiator (concensus WANt/aYY; Smale and

Baltimore 1989). In rnammals, for example, the majority of Pol II transcribed genes

have a TATA box located around 30 base pairs upstream and an initiator element

overlapping the start site of transcription. Additional conserved Pol II promoter

sequences have also been documented (Bucher 1990; Burke and Kadonaga 1996).

Functional analysis of these core promoter elements indicates that they are

somewhat functionally redundant in that each is capable of directing Pol II to

initiate transcription from an adjacent gene (Carcamo et al. 1991; O'Shea-Greenfield

and Smale 1992; Aso et al. 1994; Zenzie-Gregory et al. 1994; Colgan and Manley

1995). However, while a combination of several of these core elements, such as

both a TATA box and an initiator, enhances the efficiency of Pol II rnediated

transcription il1 vitro (Simon et al. 1988; Farnham and Means 1990; O'Shea-

Greenfield and Smale 1992; Nakatani et al. 1990; Carcamo et al. 1991) it is, for the

most part, the presence of gene-specific cis-acting regulatory sequences, located

either upstream or downstream of the start site of transcription, which

determines the physiological levels of transcription of a gene in a ceil (Struhl 1981;

McKnight and Kingsbury 1982; Guarente et al. 1984; Struhl, 1984; Zenke et al. 1986;

Simon et al. 1988; Chang and Gralla, 1993). Strikingly, many of these regulatory

sequences function even when located at a great distance ( z l kb) distal to a target

promoter. However, in the majority of cases these regulatory sequences are usually

found adjacent ( ~ 4 0 0 bp) to the core promoter elements (reviewed by Dynan and

Tjian 1985; McKnight and Tjian 1986; and Struhl 1993, 1995).

II. Sequence-Specific Activators of Transcription

A key advance in understanding how Pol II is regulated arose from studies

demonstrating that sequence-specific DNA-binding proteins interact with the cis-

acting elements found upstream of the core promoters and that these proteins

potentiate the activity of Pol II at a nearby promoter (Dynan and Tjian 19S3a,

1983b; Guarente et al. 1984; Bram and Kornberg, 1985; Giniger et al. 1985; Hope and

Struhl 1985; Dynan and Tjian 1985; Sawadogo and Roeder 1985; Briggs et al. 1986;

Jones et al. 1986, 1987; Olesen et al. 1987; Pfeifer et al. 1987). It is now apparent that

the interaction of these transcriptional activators (hereafter referred to simply as

transactivators) with particular regulatory sequences, such a s upstream activating

sequences (UASs; Struhl 1984; Olesen et al. 1987) or enhancers (Zenke et al. 1986;

Ondek et al. 1988), is a crucial event in the regulated expression of most, if not all,

messenger RNAs (reviewed by Dynan and Tjian 1985; McKnight and Tjian 1986;

and Struhl 1995).

Since their initial discovery, a large number of transactivators have been

identified from a broad range of eukaryotic organisms. Given their relative

importance to gene regulation, many of these transactivators have been subject to

a battery of biochemical and genetic tests aimed at dissecting the molecular details

of their structure-function relationship. Taken together, these studies have

highlighted certain characteristics common to transactivators in general.

First, transactivators tend to bind to their cognate DNA sequences with

high specificity and affinitv. In vitro binding studies have indicated that the

majority of transactivators bind to their cognate sequences, at least on naked

DNA, with a K d in the range of 10-9 to 10-10 M, an affinity several orders of

magnitude greater than their affinity for non-specific DNA sequences (reviewed

by Mitchell and Tjian 1989 and Morimoto 1992). Site-directed mutagenesis,

DNase 1 footprinting, and X-ray crystallographic studies have demonstrated that

transactivators interact with short DNA sequences, usually only 8 to 12 base pairs

long, through direct and specific contacts with bases in the major groove of the

DNA helix (reviewed by Mitchell and Tjian 1989 and Morimoto 1992). This ability

to selectively target transactivators to specfic DNA sequences explains to a large

extent how cells regulate Pol LI mediated transcription in a very precise (i.e. gene-

specific) manner.

While a single transactivator binding site is often sufficient to support

activated levels of transcription frorn a synthetic reporter gene (see, for example,

Diamond et al. 1990 and Segal and Berk 1991), most naturally occuring promoters

contain multiple binding sites for one or more distinct transactivators (reviewed

by Dynan and Tjian 1985; McKnight and Tjian 1986; and Struhl 1995). Such a

configuration clearly confers a greater flexibility in the replat ion of transcription.

Simultaneous binding of several transactivators to sequences upstream of a

promoter results in synergistic (Le. greater than additive) effects on transcription

(Lin et al. 1988; Courey and Tjian 1989; Carey et al. 1990; Anderson and Freytag

1991) leading to greatly elevated levels of gene expression. In certain

circumstances, this synergistic response results from cooperative binding of the

transactivators to DNA (Olesen et al. 1987; Janson and Pettersson 1990). More

generally, however, it appears to reflect an enhanced ability of the transactivators

to interact productively with Pol II and its associated transcriptional machinery

(section V) at an adjacent promoter (Lin et al. 1988; Courey et al. 1990; Lin et al.

1990; Diamond et al. 1990; Emani and Carey 1992; Seipel et al. 1992).

Most transactivators are, in tum, subject to a high degree of regulation.

Transactivators often serve as the downstream targets of signal transduction

pathways which tailor their activities to meet specific physiological requirernrnts

(reviewea by Sassone-Corsi 1992). Misregulation of transactivator function can

have dramatic consequences on eukaryotic ce11 growth and development. in

particular, deregulated transactivation is associated with a number of

developmental defects in humans.

Transactivators are regulated by a number of different molecular

mechanisms; 1) including altered levels of transcription or translation, 2) post-

translational modifications such as phosphorylation, or 3) through controlled

nuclear localization (reviewed by Falvey and Schibler 1992; Hunter and Karin

2992; Struhl 1995; and Calkhoven and Ab 1996). In certain cases, transactivators

may also be regulated through direct physical association with specific positive- or

negative-acting transcriptional cofactors (reviewed by Calkhoven and Ab 1996).

An example of the latter form of regulation is exemplified by the transactivator

GAL4 which regulates the expression of genes required for the metabolism of

galactose in budding yeast (reviewed by Lohr et al. 1995). When yeast cells are

grown in the presence of galactose (as the sole fermentable carbon source),

transcription by GAL4 is markedly induced; however, in the absence of galactose

GAL4 is maintained in an inactive state through binding of the specific inhibitor

protein, GALSO (Ma and Ptashne 1987b; Johnston et al. 1987). Similarly, the

human transactivators p53 and E2F-1 are negatively regulated by specific and

direct binding of the inhibitory proteins mdm-2 (Momand et al. 1992) and Rb

(Flemington et al. 1993) respectively.

The activation function of transactivators is mediated through a distinct

region of the protein termed the activation domain (Hope and Stmhl 1986;

Keegan et al. 1986; Ma and Ptashne 1987a, 1987b; Courey and Tjian 1988; Kadonaga

et al. 1988; Sadowski et al. 1988) which is usually hnctionally separable from the

DNA-binding portion (or domain) of the protein (reviewed by Mitchell and Tjian

1989). The separable nature of the activation domain is indicated by the fact that

the activation domains of many diverse transactivators will potentiate

transcription when fused to a heterologous DNA-binding domain, such as that of

GAL4 (see, for example, Fitzpatrick and Ingles 1989) or the bacterial protein LexA

(Brent and Ptashne 1985; Godowski et al. 1988; Lech e t al. 1988). Activation

domains can also potentiate transcription when tethered to a DNA-binding

protein through a non-covalent interaction (Ma and Ptashne 1988; Fields and

Song 1989; Ho et al. 1996) further emphasizing the independent nature of these

two functions. Thus, although DNA-binding is a prerequisite event for

transactivator function, it is not sufficient for stimulation of Pol II activity to

occur. Once tethered to a nearby promoter, the activation domain is presumed to

lie on the surface of the transactivator in a manner which allows it to interact

with a component(s) of the Pol II transcriptional machinery (section V). This

activation process must be a limiting event for each round of transcription of a

particular gene since an activation domain must be continuously tethered

upstrearn of a promoter in order for multiple rounds of transcription to occur (Ho

et al. 1996).

The activation domains of many transactivators of diverse origin are

recognizable by the presence of common structural motifs (reviewed by Mitchell

and Tjian 1989 and Krajuska 1992). For example, the activation domains of manv

transactivators of mammalian (eg. p53, E2F-l), viral ( eg . VP16), and yeast

( e g . GCN4 and GAL4) origin are rich in negatively charged or acidic arnino acids

(Hope and Struhl 1986; Ma and Ptashne 1987a; Sadowski et al. 1988; Triezenberg et

al. 1988; Fields and Jang 1990; Flemington et al. 1993), although negative charge is

not a sufficient or essential parameter of activation domain function (Cress and

Triezenberg 1991; Regier et al. 1993; Leuther et al. 1993). This observation,

combined with the fact that most transactivators function when expressed in a

variety of non-native eukaryotic cells, has led to the suggestion that many, if not

all, transactivators function through common evolutionarily conserved

mechanisms.

III. The Pol II transcription cycle

In order to criticallv evaluate the mechanisms by which transactivators

modulate the activity of Pol II, the molecular events that are basic to the process

of Pol II-mediated transcription must be considered. Promoter-dependent

transcription by Pol II can be resolved experimentaily i r l oitro into six sequential

steps which are defined operationally as (1) promoter commitment, (2) open

' (5) complex formation, (3) initiation of transcription, (4) promoter clearance

chain elonga tion, and (6) termination.

In the first step of this cycle, Pol II and its associated transcriptiona

machinery (section V) interact initimately with the core promoter elements to

form a closed preinitiation complex (Hawley and Roeder 1985, 1987; Van Dyck et

al. 1988; Buratowski et al. 1989). The DNA strands surrounding the start site of

transcription are then separated, or melted, in an ATP-dependent rnanner (Jiang

et al. 1993), thereby exposing the coding strand to the catalytic site of the

polymerase. The formation of this open complex is a prerequiste for the synthesis

of the first phosphodiester bond (Wang et al. 1992) which occurs rapidly thereafter

(Wang et al. 1992; Jiang et al. 1996). The initiation of transcription leads to a

rearrangement in the association of the transcrip tional machnery wi th the

promoter resulting in an expansion of the melted region surrounding the start

site of transcription (see, for example, Giardina and Lis 1993b). After initiation, the

enzyme either pauses stably at the 5' end of the gene (Luse and Jacob 1987;

Kerppola and Kane 1988; Krumm et al. 1995), possibly svnthesizing short

oligonucleotides reiteratively (Luse and Jacob 1987; Jiang et al. 1995), or disengages

from the promoter and proceeds to a processive mode of chain elongation

(reviewed by Bentley 1995). The cycle is completed upon transcriptional

termination, likely the result of the passage of Pol II through particular DNA

sequences (sec for example Dedrick et al. 19871, resulting in the release of the

nascent RNA transcript and dissociation of Pol 11 from the DNA.

There is experimental evidence that each of these molecular transitions

can be rate-limiting for the transcription of particular genes. For example, studies

using potassium permanganate, which reacts specifically with T-residues in

single-stranded DNA, have indicated that the initiation of transcription can be

limited bv the rates of steps leading to the formation of the open preinitiation

cornplex and melting of the promoter DNA around the start site of transcription

(Wang et al. 1992a; Wang et al. 1992b; Jiang et al. 1994, 1995). Therefore,

transactivators may stimulate transcription both by enhancing the rate of

assembly of a productive preinitiation complex as well as by stimulating the

molecular transitions which limit initiation of the transcript. In practice, certain

transactivators appear to stimulate one particular step in the transcription cycle,

whereas others appear to act at multiple steps in the overall pathway.

Several lines of evidence indicate that one key function of manv, if not all,

transactivators is to enhance the recruitment of Pol Il and its associated

transcriptional machinery to a promoter. First, one must consider that the DNA

in the nucleus of a living ce11 is wound tightly around histone proteins in the

form of nucleosomes. These nucleoprotein complexes appear to inhibit

transcription by limiting the access of the Pol LI transcriptional machinerv to the

DNA (reviewed by Wolffe 1992). For example, when located over the core

promoter elements, a nucleosome can effectively block the formation of a Pol II

preinitiation complex in aitro (Fedor et al. 1992; Workman and Buchman 1993;

reviewed by Wolffe 1992). This suggests that in order to s tirnulate transcription

from an adjacent promoter, transactivators must first overcome the repressive

effects inherent to chromatin structure. Consistent with this notion, in Z ~ ~ Z J O

footprinting studies have shown that, upon binding to DNA, a number of

transactivators destabilize the formation of nucleosomes at adjacent promoters in

a manner which likely exposes the core promoter elements for engagement by

Pol II (Schmid et al. 1992; Axelrod et al. 1993; Cavalli and Thorna 1993; Pazin et al.

1994; Svaren et al. 1994; Pazin et al. 1996). Similarly, it has also been established

that transactivators stimulate the formation of a preinitiation cornplex on DNA

templates reconstituted into chromatin-like structures irz vitro (Workman et al.

1988; Laybourn and Kadonaga 1991; Workman et al. 1991; Croston et al. 1991,1992;

Layboum and Kadonaga 1992; Pazin et al. 1994; Paranjape et al. 1995). This so-

called anti-repression function of transactivators appears to be closely linked to

the process of transcriptional activation since it requires the presence of a

functional activation domain within the transactivator.

Second, time course and template commitment studies performed in vitro

have indica ted tha t transactivators stimulate the intrinsic rate of transcrip tional

initiation by Pol II even in the presence of naked (i.e. non-chromatin) DNA

templates (Hai et al. 1988; Horikoshi et al. 1988a; Horikoshi et al. 1988b; Wang et

al. 1992a, 1992b; White et al. 1992). These studies suggest that transactivators are

required for the formation of a productive and stable promoter preinitiation

complex even in the absence of a repressive chrornatin structure. As discussed

below (section V), the assembly of a productive preinitiation complex involves

the coordinated interaction of a large number of distinct protein factors with the

promoter, suggesting there may be several different targets at this stage for

regulation b y transactiva tors.

In addition to regulating the recruitment and initiation phases of

transcription by Pol II, a growing body of evidence suggests that transactivators

also potentiate transcription of certain genes by enhancing the activity of Pol II

subsequent to its engagement with the promoter. For example, studies of the

Drosophiln heat shock genes in which Pol II was crosslinked to DNA ili i7io0

have indicated that Pol II generally becomes stalled after transcribing the first 20 to

30 nucleotides of these genes. Transcription only resumes upon binding of an

activated form of the gene-specific transactivator known as heat shock factor

(Giardina and Lis 1993a). Studies of transcriptional activation in oitro using a

different set of reporter genes has also indicated that a number of other

transactivators function by stimulating the rate of promoter clearance by Pol II

(Narayan et al. 1994; Jiang et al. 1996).

Finally, nuclear run-on experiments in yeast, human, and Xeiiopiis cells

(Akhtar et al. 1996; Marcianiak and Sharp 1991; Yankulov et al. 1994; Blau et al.

1996; Blair et al. 1996) as well as studies in cell-free systems in oitro (Kato et al.

1992; Laspia et al. 1993) have provided evidence that transactivators also markedly

stimulate N A chain elongation by Pol II in addition to their effects on initiation

and promoter clearance. In this case, it appears that transactivators enhance the

formation of a more processive f o m of the Pol II which is resistant to premature

pausing or arrest (reviewed by Greenblatt et al. 1993 and Bentley 1995). As most

mammalian genes are very large, the ability of transactivators to stimula te the

rate of chain elongation by Pol II may prove to be as vital as their effects on the

initiation of transcription.

IV. The CTD

Pol II is composed of twelve core polypeptide subunits. The largest of these

subunitç has a unique carboxy-terminal domain (CTD) which consists of an array

of highly conserved heptapeptide repeats (consensus Tyr-Ser-Pro-Thr-Ser-Pro-Ser)

that is reiterated twenty-six times in yeast (Allison et al. 1985; Nonet et al. 1987)

and fifty-two times in mammals (Corden et al. 1985; Allison et al. 1988). This

sequence is essential (Bartolomei et al. 1988; Nonet et al. 1989; Allison et al. 1988)

but is not found in the homologous subunits of RNA polymerase I and RNA

polymerase III or in the homologJ' in E. coli RNA polymerase (Allison et al.

1985, 1988). The CTD appears to be essential for either initiation of transcription

(dinucleotide-phosphodiester formation) or for promoter clearance by pol II from

different promoters iiz z~itro (Akoulitchev et al. 1995). As such, it is thought to be

involved in regulating some basic aspect of the activity of Pol II (reviewed bv

Corden and Ingles 1992).

Several observations suggest that the CTD plays a major role in regulating

the response of the Pol II transcriptional machinery to transactivators. First, the

CTD is required for Pol II-mediated transcription of a number of inducible genes

in vivo (Scafe et al. 1990; Meisels et al. 1995) and in iritro (Buermyer et al. 1992).

Second, truncation of the C m impairs the ability of certain transactivators to

stimulate transcription iit oitro (Liao et al. 1991; Okamoto et al. 1996) and in

living cells (Gerber et al. 1995; Okamoto et al. 1996). Third, variations in the

length of the CTD can either enhance or suppress the effects of mutations in the

activation domain of a transactivator (Allison and Ingles 1989).

How might the CTD mediate the effects of transactivators o n the activity of

Pol II? A possible role for CTD phosphorylation in regulating activation of Pol II

was suggested by the observation that the CTD exists in both an

unphosphorylated and a hyperphosphorylated form iri z~ii10 (reviewed by

Dahmus 1996). Furthermore, phosphorylation of the CTD occurs predominantly,

if not exclusively, in a promoter-dependent manner (Lu et al. 1992; reviewed by

Corden 1993 and Dahmus 1996). Although the precise functional consequences of

CTD phosp horylation are poorly understood, only the non-phosp horyla ted form

of Pol II appears to associate with a promoter in vitro (Lu et al. 1991; Chesnut et

al. 1992) and iri vivo (O'Brien et al. 1994). Since phosphorylation of the CTD

coincides with the transition from the initiation of transcription to chain

elongation (Payne et al. 1989; O'Brien et al. 1994), one possibiiity is that

transactivators enhance the efficiency of promoter clearance by Pol II by somehow

inducing phosphorylation of the CTD. Consistent with this notion, it has been

shown that pharmacological inhibitors of CTD phosphorylation reduce the rate of

promoter clearance by Pol II as well as transcriptional activation by a number of

transactivators in vivo (Braddock et al. 1991; Marcianiak and Sharp 1991;

Giardina and Lis 1993; Yankulov et al. 1995). Furthermore, irz vii70 crosslinking

and irnrnunohistochemical s taining s tudies have suggested that it is

predominantly the hyperphosphorylated form of Pol II which mediates chain

elongation (Cadena and Dahmus 1987; Weeks et al. 1993). Therefore, the ability of

transactivators to stimulate phosphorylation of the CTD may also contribute to

the formation of a more processive elongation complex (reviewed by Bentley

1995).

V. The Pol II Transcnptional Machinery

The development of cell-free systems capable of accurate promoter-

dependent transcription by pol II in z~itro (Weil et al. ;979; Matsui et al. 1980;

Manley et al. 1980; Dignam et al. 1983; Sawadogo and Roeder 1985; Lue et al. 1987;

Shapiro et al. 1988; Lue et al. 1989; Chasman et al. 1989; Kamakaka et al. 1991;

Woontner et al. 1991; Flanagan et al. 1992) has paved the way For a more extensive

biochemical analysis of the process of transcriptional activation. For example,

chrornatographic fractionation of these extracts has led to the discovery of

multiple accessory protein factors which are either essential for transcription by

Pol II or which modulate its response to transactivators. Many of these accessory

factors have been evolutionarily conserved, providing additional evidence that

the mechanisms b y which transactivators function have also been conserved.

Finally, the ability to reconstitute Pol II-mediated transcription i rr oiti-u using a

fairly well defined set of proteins has led to models detailing how transactivators

function. In the next section, 1 highlight various aspects of the Pol II

transcriptional machinery which appear relevant to our understanding of the

process of transcriptional activation. For a more comprehensive review of the

structure and function of the various components of the Pol II transcriptional

machinery, the reader is directed to revieivs by Conaway and Conaway 1993;

Zawel and Reinberg 1993, 1995; and Orphanides et al. 1996.

i. The General Transcription Factors

Purified forms of the core Pol II enzyme are capable of transcribing a nicked

DNA template in a non-specific marner in vitro (Roeder 1974; Matsui et al.

1980). However, accurate promoter-dependent transcription by Pol II requires at

least five additional protein factors, known as TFIID (Matsui et al. 1980; Reinberg

and Roeder 1987; Nakajima et al. 1988), TFIIB (Reinberg and Roeder 1987; Ha et al.

1991; Sayre et al. 1992), TFIIE (Reinberg and Roeder 1987; Okhuma et al. 1990;

Peterson et al. 1991; Feaver et al. 1994a), TFIIF (Sopta et al. 1985; Burton et al. 1986,

1988; Flores et al. 1989; Sopta et al. 1989; Finkelstein et al. 1992; Henry et al. 1992),

and TFTIH (Flores et ai. 1992; Feaver et al. 1992; Sayre et al. 1992). These factors are

collectivelv referred to as the general transcription factors (GTFs). During the

assembly of a productive preinitiation compIex, the GTFs associate with Pol II and

the core promoter elements through an extensive network of protein-protein and

protein-DNA interactions. Biochemical studies based on nuclease protection,

electrophoretic mobility shift, and DNA-template challenge assays, as well as iiz

o i h mutagenesis of individual GTFs, have allowed for a detailed dissection of

many of these intermolecular interactions (reviewed bv Zawel and Reinberg 1995;

Roeder 1996; Orphanides et al. 1996). These studies have, in turn, suggested that

transactivators potentiate transcription, at least in pari, by either stimulating the

rate of association of particular GTFs with the promoter or by inducing specific

qualitative changes in their activity subsequent to the formation of the

preinitiation complex.

a. Initiation Factor TFIID

TFIID plays a key role in the initiation of transcription by Pol II since it

interacts directly and extensively with the core promoter sequences and nucleates

the subsequent formation of a preinitiation complex at most, if not all, cellular

promoters (Sawadogo and Roeder 1985; Nakajima et al. 1988; van Dyke et al. 1988;

Nakatani et al. 1990; Chiang e t al. 1993; Purnell et al. 1994; K a u h a m and Smale

1994; Sypes and Gilmour 1994; Burke and Kadonaga 1996; reviewed by Greenblatt

1991; Burley and Roeder 1996). Importantly, several observations suggest that

TFIID is a critical target of many transactivators. First, the interaction of TFDD

with the promoter can be a rate-limiting event for the initiation of transcription

from a number of different cellular and viral genes in i7itr0 (Abmayr et al. 1988;

Horikoshi et al. 1988a and 1988b; Van Dyke et al. 1989; White et al. 1990;

Lieberman and Berk 1991; Wang et al. 1992). Second, a large number oi diverse

transactivators can either stabilize the binding of TFIID to core promoter

sequences or alter the conformation of the TFIID-promoter complex in a manner

that correlates directly with the ability of transactivators to activate transcription

(Abmayr, et al. 1988; Horikoshi et al. 1988a and 1988b; White et al. 1990; Lieberman

and Berk 1991; Wang et al. 1992). As the association of TFIID with the core

promoter elements precludes the formation of a nucleosome complex (reviewed

by Roeder and Burley 1995), the ability of transactivators to selectively recruite

TFIID to a promoter also expiains how transactivators might stimulate

transcription from genes embedded in chromatin.

b. The TATA-Binding Protein

TFIID is a large protein complex composed of a central TATA-binding

subunit (TBP) in association with ten or more TBP-associated factors or TAFs

(Dynlacht et al. 1991; Tanese e t al. 1991). As such, the interaction of transactivators

with any one of these subunits might, in principle, be important for

transactivator-mediated recruitrnent of TFIID to a promoter. However, a number

of observations implicate TBP as the principle target in TFIID of most

transactivators.

First, TBP is the only subunit of TFIID which is absolutely required for the

initiation of transcription iil vitro (reviewed by Burley and Roeder 1996). Second,

overexpression of TBP in human cells greatly enhances the transcription of

certain cellular genes (Colgan and Manley 1992) and the response of the Pol II

transcriptional machinery to exogenous transactivators (see, for example,

Sadovski e t al. 1995). Third, the interaction of TBP with the TATA elernent can be

a rate-limiting step for the initiation of transcription of certain genes, both i n

vitro and in z~iiw, and this step can be accelerated by transactivators (Lieberman

and Berk 1991; Klein and Struhl 2994). Fourth, it has been found that the

activation domains of a large number of cellular and viral transactivators are able

to interact directly and specifically with TBP in vitro (see, for example, Stringer et

al. 1990; Lee et al. 1991; Lieberman and Berk 1991; Liu et al. 1993; Truant et al. 1993;

Kashanchi et al. 1994; Melcher and Johnston 1995, Wu et al. 1996). in particular,

studies in the Ingles and Greenblatt laboratory have shown direct binding of the

acidic activation dornain of the herpes viral protein VP16 to yeast TBP (Stringer et

al. 1990) and of the glutamine-rich activation domains of Spl to human TBP

(A. Emili, M.Sc. Thesis 1994; Emili et al. 1994). The biological significance of the

interaction of transactivators with TBP is strengthened by the observation that

mutations in either the activation domain of the transactivator (Ingles et al. 1991)

or in TE3P (Kim et al. 1994; but see Tansey and Herr 1995) which abrogate this

interaction also compromise activation of transcription.

How might the contact between transactivators and TBP affect

transcription? One plausible answer stems from the fact that the association of

TBP with the minor grooïe of the DNA helix results in an extreme structural

distortion of the promoter (Kim et al. 1993; Nikolov et al. 1996; reviewed by

Burley and Roeder 1996). Thus, although TBP c m bind to concensus TATA

elements on naked DNA templates in uitro (Horikoshi et al. 1989; Hahn et al.

1989a, 1989b; Peterson et al. 1990), it mav be that transactivators are required to

stabilize the binding of TBP to weak, non-concensus TATA elements or to TATA

elements embedded in chromatin. Consistent with this notion, it has been shown

that fusing TBP directly to a sequence-çpecific DNA-binding protein c m result in

activated levels of transcription in oioo in the absence of a bone fide activation

domain (Chatte rjee and Struhl 1994, Klages and Strubin 1994; Xiao et al. 1995).

Furthermore, it has also been shown that mutations in the DNA-binding surface

of TBP which impair its ability to interact with DNA also block the abilitv of

transactivators to potentiate transcription (Kim et al. 1994; Arndt et al. 1995; Lee

and Struhl 1995).

As TBP interacts with a variety of other transcriptional components

(reviewed by Burley and Roeder 1985), such as the TAFs (Dynlacht et al. 1991;

Tanese et al. 1991), TFIIB (Buratowski and Zhou 1992), the regulatory protein

TFIIA (Buratowski and Zhou 1992; section V), and the CTD domain of the largest

subunit of Pol II (Usheva et al. 1992), direct contact by transactivators might also

modulate the range of protein-protein interactions mediated by TBP at a

promoter. Consistent with this hypothesis, it has been shown that transactivators

can stimulate the rate of association of both TFEB and TFIIA with the TBP-

promoter cornplex in vitro (Lin and Green 1991; Lieberman and Berk 1991;

Sundseth and Hansen 1992). Furthermore, the ability of transactivators to interact

directly with TBP may also reverse the effects of specific repressors of

1-20

transcription, such as Dr1 (Meisteremst and Roeder 1991; Inostroza et al. 1992;

Yeung et al. 1994; Kim et al. 1996), Mot1 (Auble and Hahn

and HMGl (Ge and Roeder 1994), which appear to inhibit

preinitiation complex by interacting directly with TBP.

1993; Auble et al. 1994),

the formation of a

c. The TBP-Associated Factors

The TAF subunits of TFDD also appear to regulate the assemblv of

productive preinitiation complexes at certain promoters, either by making direct

contact with specific promoter DNA sequences (Pugh and Tjian 1991; Kaufmann

and Smale 1994; Martinez et al. 1994; Pumell et al. 1994; Sypes and Gilmour 1994;

Verrijzer et al. 1994; Hansen and Tjian 1995; Verrijzer et al. 1995; Burke and

Kadonaga 1996) or by stabilizing the interaction of other components of the Pol II

transcription machinery (Goodrich and Tjian 1993; Aso et al. 1994; Lieberman and

Berk 1994; Martinez et al. 1994; Chi et al. 1995; Ruppert and Tjian 1995). TBP can

support a basal level of activator-independent transcription in reconstituted ce11

free transcription systems iri z~itro (Buratowski et al. 1988; Horikoshi et al. 1989;

Hahn et al. 1989a, 1989b; Hoey et al. 1990; Peterson et al. 1990), but only the TFIID

complex supports activated levels of transcription in both human and Drosophiln

cell extracts (Hoey et al. 1990; Peterson et al. 1990; Pugh and Tjian 1990; Dvnlacht et

al. 1991; Tanese et al. 1991; Zhou et al. 1992). This observation suggests that the

TAFs, at least in humans and Drosopliiln, are critical determinants in the process

of transcriptional activation. Yeast hornologs of the mamrnalian TAFs have been

identified (Poon et al. 1995; Reese et al. 1995), illustrating, once again, the

remarkable conservation of the basic transcriptional machinery.

It has been suggested that, as with TBP, the TAFs might serve as important

targets of transactivators (reviewed by Verrijzer and Tjian 1996). Indeed, a

number of in iitro binding studies have provided evidence for specific and direct

transactivator-TAF interactions (Goodrich et al. 1993; Hoev et al. 1993; Chen et al.

1994; Chiang and Roeder 1995). The ability of various combinations of

transactivators and TAF subunits to interact in vitro has correlated remarkably

well with the capacity to reconstitute activated levels of transcription i r i i7itr0

(Chen et al. 1994; Thut et al. 1995; Sauer et al. 1995a, 1995b). As such, it was

assumed that the TAFs were biologically relevant targets of transactivators in

oizo (reviewed by Verrijzer and Tjian 1996). Recent studies in yeast have cast

doubt on this notion. It has been shown that activated levels of transcription can

be achieved i r l aitro in the absence of TAFs with some purified forms of the yeast

Pol II transcriptional machinery (Kelleher et al. 1992; Koleske and Young 1994;

Kim et al. 1994). Furthermore, and more conclusively, it was found that

functional inactivation or depletion of the TAFs in yeast does not impair the

ability of a number of different transactivators to stimulate transcription in

vivo(Wa1ker et al. 1996; Moqtaderi et al. 1996; Apone et al. 1996). Thus, there is as

yet no conclusive evidence for a physiological role for TAFs as essential targets of

transactivators. Nonetheless, that being said, it is also unlikely that TBP is the

only important target of transactivators. Indeed, biochemical and genetic studies

indicate that transactivators function by modulating the activity of a number of

other components of the Pol II transcriptional machinery (see below).

d. TFIIB

TFIIB is a monomeric GTF that associates directly with Pol II and the

TBP/TFIID-promoter complex (Ha et al. 1991,1993; Nikolov et al. 1995). In this

marner, TFIIB appears to bridge the subsequent entry of Pol LI to the promoter

(Buratowski et al. 1989; Ha et al. 1991, 1993). TFIIB is oriented through contacts

with DNA sequences surrounding the TATA-box (Coulombe et al. 1992; Nikolov

et al. 1995; reviewed by Roeder 1996) and is involved in the selection of the start

site of îranscription (Pinto et al. 1992; Li et al. 1994) presumably by positioning the

active site of Pol II in relation to the core promoter sequences. The ability of TFFIIB

to interact with TBP also appears to be essential for transcriptional activation in

uiuo (Bryant et al. 1996). The association of TFIIB with the TBP/TFIID-promoter

complex can be, under certain experimental conditions, a rate-limiting step for

initiation of transcription i r i nitro (Lin and Green 1991a; Choy and Green 1993),

therefore, TFIIB may be an important target for regulation by transactivators.

Consistent with this notion, it has been shown that certain transactivators can

stabilize the association of TFIIB with a promoter preinitiation complex in zitl-o

(Lin and Green 1991; Sundseth and Hansen 1992; Choy and Green 1993; Kim et al.

1994; Kim and Roeder 1994; Chi et al. 1995). Since TFIIB appears to dissociate from

the prornoter following initiation of the transcript (Zawel et al. 1995), i t rnav also

be that transactivators hasten the reincorporation of TFIIB into the preinitiation

complex during succesive rounds of transcription. However, since a large molar

excess of TFIIB does not markedly stimulate the efficiency of initiation i i i oitro

(White et al. 1992), iransactivators need not solely function by enhancing the rate

of association of TFIIB with a promoter. For example, transactivators may also

change the conformation of TFIIB (Roberts and Green 1994) such that the

association of the remaining GTFs with the promoter is stimulated (Choy and

Green 1993). One or more of these effects may reflect the ability of transactivators

to interact directly with TFIIB (Lin and Green 1991b; Roberts et al. 1993; Colgan et

al. 1993; MacDonald et al. 1995; Sauer et al. 1995; Wu et al. 1996) although the

functional significance of this binding has been challenged (Goodrich and Tjian

1993; Walker et al. 1993; Gupta et al. 1996).

e. TFIIE, TFIIF, and TFIIH

TFIIE and TFIF are two multisubunit GTFs that also play an important

role in regulating transcriptional initiation by Pol II. Like T'FIIB, TRIE and TFIIF

interact directly with Pol II and stabilize its interaction with the promoter

(Reinberg and Roeder 1987; Sopta et al. 1985; Burton et al. 1986; Flores et al. 1989;

Killeen 1992; Maxon et al. 1994; Bushnell et al. 1996), possibly through contacts

with the non-phosphorylated f o m of the CTD (Maxon et al. 1994; Kang and

D a h u s 1995). Photo-crosslinking studies have indicated that both factors contact

the DNA helix immediately upstream of the start site of transcription (Robert et

al. 1996). I t has been suggested that the TFIIE-F-DNA contacts assist in localizing

the catalytic site of Pol II in relation to the promoter and stabilize the single-

stranded DNA region around the start site of transcription (Leuther et al. 1996). A

number of stiidies also suggest that both of these factors stimulate the unwinding

of the DNA helix around the start site of transcription (Goodrich and Tjian 1994;

Pan et al. 1994; Holstege et al. 1995; Holstege et al. 1996). In addition to this role in

the initiation of transcription, TFIIE and TFLIF are also required for efficient

prornoter clearance by Pol II (Chang et al. 1993; Goodrich and Tjian 1994; Pan et al.

1994; Tan et al. 1995). Therefore, the ability of certain transactivators, such as Jun

and Fos (Martin et al. 1996), serum response factor (Zhu et al. 1994; Joliot et al.

1995), and a subset of homeodomain-containing proteins (Zhu and Kuziora 1996),

to make physical contacts with either TFIIE or TFIIF may be critical for their ability

to stimulate the earliest stages of transcription by Pol II.

TFIIH is another appealing target for regdation by transactivators since it

has a number of intrinsic enzymatic activities. TFIIH exhibits protein kinase,

DNA-dependent ATFase, and Am-dependent DNA helicase activities (Feaver et

al. 1991; Fisher et al. 1992; Lu et al. 1992; Serizawa et al. 1992; Feaver et al. 1993;

Schaeffer et al. 1993; Roy et al. 1994). Consistent with its broad enzymatic

activities, TFIIH is a large multisubunit protein complex (Feaver et al. 1991, 1993;

Schaeffer et al., 1993; Qiu et al., 1993; Draplun et al. 1994; Svestrup et al. 1994, 1995).

The helicase activity of TFIIH appears to be critical for melting of the promoter

region around the start site of transcription (Timmers, 1994; Tantin and Carey

1994; Pan et al. 1994; Holstege et al. 1996; see also Parvin and Sharp 1993) and

likely accounts for the strict requirement for ATP P-y bond hydrolysis during the

initiation of transcription (Bunick et al. 1982; Jiang et al. 1993; Tirnmers 1994;

Holstege et al. 1996). The protein kinase activity of TFIIH is also essential for

transcription by Pol II (Feaver et al. 1991; Feaver et al. 1993; Lu et al. 1992; Serizawa

et al. 1992; Rov et al. 1994; Feaver et al. 1994b; Svejstrup et al. 1994, 1993). Of

particular significance is the fact that TFIIH can specificaily phosphorvlate the

CTD in aitro (Feaver et al. 1991; Lu et al. 1992; Serizawa et al. 1992; Feaver et al.

1993, 1994b). Indeed, TFIIH is likely the principle CTD kinase in the ce11 since

functional inactivation of the catalytic subunit of the yeast TFIIH kinase abolishes

CTD phosphorylation bi i~ii70 (Cismowski et al. 1995; Valay et al. 1995). nerefore,

that transactivators such as VP16 (Xiao et al. 1994), p53 (Xiao et al. 1994; Leveillard

et al. 1996), HIV-1 Tat protein (Blau et al. 1996), and Hepatitis B virus protein HBx

(Qadri et al. 1996) interact directly with T F W may mediate their stimulatory

effects on transcription by enhancing the rate of formation of the open

preinitiation complex as well as the rate of promoter clearance and chain

elongation by pol II. Consistent with this notion, it was shown that inactivation

of the TFIIH kinase impairs the ability of transactivators to stimulate

transcription in yeaçt (Akhtar et al. 1996).

TFIIE and TFIIF mav also be important targets for mediating the effects of

transactivators on chain elongation by Pol II. For example, transactivators may

inhibit the activity of a Cm-specific phosphatase associated with TFIIF (Chambers

et al. 1995; Chambers and Kane 1996; Archambault et al. 1996, manuscript

submitted). Furthermore, P I I E stimulates TFIIH-mediated phosphorylation of

the CTD in vitro (Lu et al. 1992; Ohkuma and Roeder 1994) and TFIIF remains

associated with Pol II during chain elongation (Zawel et al. 1995). Therefore, the

ability of transactivators to modulate the activities of either of these GTFs, as well

as the kinase activity of TFIIH, may lead to the common result of a more

processive, hyperphosp horylated form of Pol II. Consistent with this notion, i t

has been shown that TFIIF modulateç the ability of Tat to enhance chain

elongation by Pol II irt i~ i t ro (Kato et al. 1992).

ii. Transcriptional Cofactors

Although the evidence presented so far suggests that transactivators

communicate directly with components of the general transcriptional machinery,

such interactions may not be sufficient for activation to occur in z~iao. A number

of biochemical and genetic observations indicate that the activation function of

transactivators is critically dependent on the activity of a number of accessory

cofactors which are distinct from the GTFs. ïhese cofactors represent a variety of

different biological activities which O ften h c t i o n in a cell-specific, gene-specific,

or even transactivator-specific marner. While the discovery of these cofactors is

not inconsistent with a central role for direct contact between transactivators and

the GTFs, it does imply that the regulation of transcription is a much more

complica ted process than initially anticipated.

a. The Mediator

Yeast cells encoding a partially truncated CTD grow slowly and are

temperature- and cold-sensitive (Dynan and Tjian 1953; Allison et al. 1958). These

phenotypes probably reflect defects in the transcription of certain essential genes.

Extragenic suppressors of yeast strains bearing a partially deleted CTD have been

isolated (Nonet et al. 1989; Koleske et al. 1992; Thompson et al. 1993). The protein

products of these genes, known as the SR& are essential for transcription of

most, if not all, protein-coding genes in oiuo (Thompson and Young 1995).

Al1 nine SRB gene products identified to date are present within a large protein

complex, termed the mediator, which appears to interact directly with the CTD of

Pol II (Kelleher et al. 1990; Thornpson et al. 1993; Kim et al. 1994; Li et al. 1995).

The mediator complex is thought to play a key role in the activation of

transcription for several reasons. First, the mediator complex potentiates

transcriptional activation when added to crude yeast ce11 extracts (Kelleher et al.

1990) or to more highly purified forms of the yeast Pol II transcriptional

machinery (Kim et al. 1994). Second, the mediator complex enhances TFIIH-

dependent phosphorylation of the CTD il1 vitro (Kim et al. 1994). Furthermore,

the mediator complex includes a number of additional protein factors, such as

GAL11, SIN4, and RGRl (Kim et al. 1994; Li et al. 1995; Song et al. 1996), which

have been implicated genetically in the regulation of transcription i ~ z i 7 i i j 0

(Himmelfarb et al. 1991; Sakai et al. 1990; Jiang and Stillman 1992; Chen et al. 1993;

reviewed by Bjorklund and Kim 1996). Since several mammalian homologç of

the SRB gene products have recently been identified in higher eukaryotes (Tassan

et al. 1995; Chao et al. 1996; Leclerc et al. 1996; Maldonado et al. 1996; Rickert et al.

1996), it is likely that the role of the mediator complex in the regulation of Pol U

has been conserved throughout evolution.

Biochemical studies on the yeast SRB proteins has revealed several clues as

to their function. For example, analysis of transcription i ~ i vitro using ce11

extracts derived from SRB mutant yeast strains suggests that the SRB proteins

contribute to the formation of a stable Pol II preinitiation cornplex (Koleske et al.

1992). Similarly, the SRBZO and SRB71 gene products have been shown to forrn a

protein kinase complex cvhich can selectively phosphorylate the CTD iii vitro

(Liao et al. 1995). This observation suggests that an interaction of transactivators

with one or more components of the mediator may lead to enhanced

phosphoryla tion of the CTD. Consistent with this notion, the ability of GAL4 to

activate transcription i n i~iiio is impaired in strains bearing a deletion of

S R B l O / l I genes while extracts from these strains exhibit reduced levels ot CTD

phosphorylation in vitro (Liao et al. 1995; Kuchin et al. 1995). Interestingly,

sequence analvsis of the S R B S , SRB9, SRBIO, and SRBZI gene products 1x1s also

indicated that they are identical to the transcriptional repressors SSN5, SSNZ,

SSN3, and SSN8, respectively (Kuchin et al. 1995; Song et al. 1996). This

observation suggests that transactivators may also stimulate transcription, at least

in part, by counterbalancing the transcriptional inhibitory properties which

appear to be associated with the mediator complex.

b. The SWIISNF Complex

Another transcriptional coactivator that has been particularly well

characterized in yeast is the SWI/SNF complex. This large multisubunit complex

con tains at least six gene products

1 -28

which are required for efficient activation of

many genes in both yeast and humans (Peterson et al. 1994; Caims et al. 1994;

Khavari et al. 1994; Caims et al. 1996; Wang et al. 1996a, 1996b; reviewed by

Peterson 1996 and Kingston et al. 1996). Loss-of-function mutations in most of the

SWI/SNF gene products result in impairment of transactivator function in yeast

(Neigeborn and Carlson 1984; Peterson and Herskowitz 1992; Laurent and Carison

1992; Yoshinage et al. 1992; Cairns et al. 1996). Since this transcription defect can be

partially suppresçed by mutations in, or lowered levels of, the core histone

proteins (Kruger et al. 1995; reviewed by Peterson 1996 and Kingston et al. 1996),

the SWI/ÇNF complex might be involved in mediating chromatin

reorganization by transactivatorç at promoters. This notion has been

substantiated to a large degree by comparative analysis of the chromatin structure

in wild type and SWI/SNF mutant yeast strains (Hirschorn et al. 1992). Xlso,

highly purified forms of the SWI/SNF complex have been shown to disrupt

nucleosome structure in an ATP-dependent marner i>i oitro (Owen-Hughes et al.

1996; Wang et al. 1996a) and can facilitate the binding of GTFs, such as TBP, to

chromatin templates (Imbalzano et al. 1994; Wang et al. 1996b). One logical

extension of these observations is that transactivators remodel the chromatin

structure at a promoter by actively recruiting the SWI/SNF complex.

c. Adaptors

Overexpression of a strong transactivator results in a generalized

impairment of transcription and a reduced ce11 growth rate (Gill and Ptashne

1988; Berger and Ptashne 1990; Berger et al. 1992; Melcher and Johnston 1996). This

transactivator mediated toxicity has been dubbed "squelching" (Gill and Ptashne

1988). Suppressors of squelching in yeast include mutations in several genes, such

as ADAZ, ADA3, ADAS, and GCN5, which appear to modulate the efficiency of

transcriptional activation in vivo (Berger et al. 1992; Pina et al. 1993; Brand1 et al.

1996; Marcus et al. 1996). As the phenotypes associated with double mutants in

this gene family are the same as those of single mutants (Marcus et al. 1994, 1996),

the protein products of these genes appear to be involved in the same

biochemical pathway. Consistent with this notion, the ADAZ, ADA3 and GCN5

gene products have been shown to interact as a stable complex (Marcus et al. 1994;

Horiuchi et al. 1995; Candau and Berger 1996).

What might be the function of this complex? One model invokes an

adaptor function for the complex in linking transactivators to components of the

Pol II general transcriptional machinery (reviewed by Guarente 1995). Consistent

with this model, the ADAî protein has been shown to interact directly with both

the activation domain of the transactivator VP16 as well as with TBP il1 zitro

(Silverman et al. 1994; Barlev et al. 1995). Recent studies have also implicated the

complex as a transcription-coupled histone acetyltransferase (Brownell et al. 1996;

Kuo et al. 1996). Acetylation of the amino-terminal tails of the core histones is

thought to induce a configuration change in nucleosome structure in a manner

which enhances the accessibility of a prornoter to the Pol II transcriptional

machinery (reviewed by Brownell and Allis 1996). Therefore, it is possible that

transactivators target chromatin disruption in a gene-specific manner bv directing

both this complex, as well as the SWI/SNF complex, to a particular promoter

region.

A number of adaptor-like coactivators have also been identified in human

cells. One of the best studied is the human CREB-binding protein, CBP, and a

related homolog p300, which are large nuclear proteins exhibiting some sequence

homology to ADAZ (Chrivia et al. 1993; Lundblad et al. 1995). CBP/P300 appear to

integrate a number of intracellular and extracellular sigalling pathwavs with the

transcriptional machinery. For example, both CBP and P300 interact in a ligand-

dependent rnanner with many of the nuclear hormone receptors (Chackravarti et

al. 1996) and in a phosphorylation-dependent manner with the transactivators

Mvc and CREB (Chrivia et al. 1993; Kwok et al. 1994; Lundblad et al. 1995). This

interaction appears to be essential for the activation of transcription by klyc and

CREB. Although the mechanisms by which either CBP or p300 potentiate

transcriptional activation are unclear, it may involve direct contact of either of

these proteins with one or more components of the general transcriptional

machinery. Consistent with this notion, it has been shown that activated forms of

CBP and p300 can mediate the association of transactivators with the Pol II

transcriptional machinery i i l uitro (Kee et al. 1996). CBP/p300 interact with the

histone acetvl transferase P/CAF (Yang et al. 1996) suggesting that the complex

may play a role in histone acetylation analogous to that of the ADA adaptor

complex.

d. TFIIA

TFIIA is another well characterized coactivator which has been identified

in yeast, humans, and Drosophih . TFIIA interacts directlv with TBP (Reinberg et

al. 1987; Buratowski et al. 1989; Hahn et al. 1989) and stabilizes the interaction of

both TTB and TFIID with the TATA box (Buratowski et al. 1989; Hahn et al. 1989;

Cortes et al. 1992; Sun et al. 1992; see also Geiger et al. 1996 and Tan et al. 1996).

Alrhough TFIIA was initially categorized as a general transcription factor

(Reinberg et al. 1987; Buratowski et al. 1989; Maldonado et al. 1990; Ranish and

Hahn 1991; Cortes 1992; Flores et al. 1992), it has since been shown to be

dispensible for basal (Le. activator-independent) transcription in vitro (Ma et al.

1993; Sun et al. 1994; Yokomori et al. 1994). Instead, several observations suggest

that TFIIA is involved in transactivator function.

First, the addition of recombinant TFIIA greatly stimulates the levels of

activa ted transcription in reconstitu ted in uitro transcription systems (Ma et al.

1993; Ozer et al. 1994; Sun et al. 1994; Yokomori et al. 1994; Kang et al. 1995; Ozer et

al. 1996). Second, the formation of the TFIIA-TBP/TFIiD-promoter complex can be

a rate-limiting step in the initiation of transcription in ~ i t r o which is markedly

enhanced by many transactivators (Lieberman and Berk 1994; Lieberman 1994;

Wang et al. 1994; Chi et al. 1995). This enhancement may, in certain cases, reflect a

direct interaction of a transactivator with TFIIA (Kobayashi et al. 1995; Clemens et

al. 1996). Third, it was found that mutations in either TBP or TFIIA that impair

their ability to interact iil i~i t ro dramatically reduce activation of transcription iii

oitro and i i i i~ii70 (Ozer e t al. 1994; Stargell and Struhl 1995; Bryant et al. 1996;

Ozer et al. 1996; Stargell and Struhl 1996). The importance of this interaction is

emphasized by the fact that fusion of the small subunit of TFIW to one such TBP

mutant almost fully restores transcriptional activation in yeast cells (Stargell and

Struhl 1995). Finally, the ability of transactivators to po ten tiate initiation is greatly

enhanced in the presence of a number of additional, albeit less well characterized,

transcriptional cofactors, such as PC4 (Ge and Roeder 1994; Kretzschmûr et al.

1994; Kaiser et al. 1995) and HMG-2 (Shykind et al. 1995), that appear to stimulate

the formation of the TFIIA/TFIID promoter complex.

In addition to stabilizing the association of TBP/TFIID with DNA, TFIIA

may also play another role in the activation of transcription. For example, TFIIA

mutants have been isolated which are defective as transcriptional coactivators

even though they retain the ability to bind TBP (Ozer et al. 1996; Stargell and

Struhl 1996). In this case, the role of TFIIA may be to modulate the interaction of

TFIID with certain promoters (Oelgeschlager et al. 1996; Chi and Carey 1996) in a

manner which stimulates the association of the rest of the general transcriptional

machinery with the DNA.

e. Eiongation Factors

While most of the transcrip tional cofactors characterized to date potentia te

transactivator-mediated stimulation of initiation by Pol II, it is even possible that

other transcriptional cohctors function by enhancing the ability of transactivators

to stimulate chah elongation by Pol II (reviewed by Reines et al. 1996). For

example, the human elongation cofactor SI11 (also known as Elongin) enhances

the catalytic rate of chah elongation by suppressing pausing by Pol II at many sites

along a DNA template (Bradsher 1993a, 1993b; Aso et al. 1995; Takagi et al. 1995).

Therefore, one may speculate that SI11 is somehow linked to the process of

transactivator-mediated formation of more processive Pol II elongation

complexes. Interestinglv, SI11 appears to be negatively regula ted through direct

binding of the product of the von Hippel-Lindau (VHL) tumor suppressor gene

(Duan et al. 1995). Therefore, it appears likely that the ability of transactivators to

potentiate chain elongation by Pol 11 is regulated through a network of positive

inputs by accessory elongation cofactors such as SI11 and by negative inputs from

specific repressors of transcriptional elongation such as VHL.

VI. Models of Transcriptional Activation

Order-of-addition experiments (see, for example, Van Dyke et al. 1988 and

Buratowski et al. 1989; reviewed by Zawel and Reinberg 1993, 1995 and Roeder

1996) have led to the view that the initiation of transcription results from the

sequential association of the GTFs and Pol II with a promoter in a series of steps

coordinated bv transactivators. In the classic multi-step model for activation of

Pol II transcription (Figure IA), a gene-specific transactivator is thought to hasten

the formation of a Pol II prornoter preinitiation complex by stimulating several

different rate-limiting intermediates in the overall assembly pathway. This

notion is supported by the observation that transactivators bind directly, at least

in ~ i t r o , to many distinct components of the Pol II transcriptional machinery

(section V). One prediction arising from this model is that each interaction

mediated bv an transactivator is likely to be crucial for the assembly of a

productive preinitiation complex at a given promoter.

This view of the assembly of the preinitiation complex as a step-wise

process has been challenged recently by the discovery of large cellular complexes

consisting of Pol II in stoichiometric association with many (Thompson et al.

1993; Kim et al. 1994; Chao et al. 1996; Maldonado et al. 1996; Wilson et al. 1996) or

al1 (Ossipoiv et al. 1996; Pan et al. 1996, subrnitted) of the general transcription

factors (for a review see Emili and Ingles 1995 [Appendix] and Halle and

Meisterernst 1996). The discovery of these Pol II holoenzyme complexes suggests

that the manv studies whic1.i have detaiied the ordered recruitment of Pol II and

the GTFs to a promoter merely reflect the catalog of individual pro tein-protein

contacts that occur within the context of a large preassembled Pol II transcription

factory. This notion leads to an alternative, albeit speculative, view that

transactivators recruit the complete Pol II transcriptional apparatus to a promoter

in a single step (Figure 1B). This model allows a certain flexibility in the specificity

of interactions between transactivators and the GTFs needed to influence the

initiation of transcription. Furthermore, the ability of transactivators to bind

cooperatively to a Pol II holoenzyme complex could also account for the

TATA + n-lv; I

0

TATA

TATA

t

Stepwise Recruitrnent

TATA 7 4

Holo-Pol II Recruitment

Figure 1. Two views of the steps involved in the activation of initiation of

transcription at a Pol II promoter. A. A transactivator is shown stimulating, in a

step-wise rnanner, the recruitment and/or the activity of individual components

of the Pol II transcrip tional machnery. This mode1 incorporates the reported

interactions of transactivators with the GTFs TFIID, TFIIB, and TFIIH. B. An

activator makes multiple contacts with different surfaces of a Pol II holoenzyme

complex and brings together, in one step, the complete Pol II transcriptional

machinery and the promoter DNA. The holoenzyme complex shown includes

the components of the mediator (ie the SRBs and GALII) and ail of the GTFs,

even though some of the latter have usually been separated from Pol II during

purification.

synergistic effects on transcription that are observed with multiple promoter-

bound transactivators.

Several observations support the view that transactivator-mediated

recmitment of the holoenzyme to promoters is an important pathwav for

activation of Pol II. First, a substantial portion of the cellular pool of GTFs are

stably associated with Pol II in vivo (Kim et al. 1994; Koleske and Young 1994;

Ossipow et al. 1995; Maldonado et al. 1996; Pan et al. 1996, submitted). Second,

purified Pol II holoenzyme complexes interact specifically with transactivators iil

vitro (Hengartner et al. 1995). Third, most of the Pol II holoenzyme complexes

isolated to date contain a significant fraction of many of the accessory

transcriptional cofactors, such as the mediator and the SWI/SNF cornplex, which

influence the process of transcriptional activation in z ~ i m (Kim et al. 1994;

Koleske and Young 1994; Chao et al. 1996; Maldonado et al. 1996; Wilson et al.

1996; Pan et al. 1996, submitted). Fourth, highly purified forms of the Pol II

holoenzyme complex can mediate a partial response to transactivators iii vitro

(Koleske and Young 1994; Kim et al. 1994; Hengartner et al. 1995; Pan et al. 1996,

submitted). Taken together, these observations suggest that a preassembled

holoenzyme complex is the form of the Pol II transcriptional machinery which is

responsive to transactivators in a cell.

Additional evidence supporting ':bis mode1 has stemmed from the

observation that yeast strains bearing a point mutation in the GALl 1 component

of the Pol II holoenzyme are able to support activation by a GAL4 derivative

lacking a boiie fine activation domain (Himmelfarb et al. 1990; Barberis et al.

1996). This mutation aliowed GALll to interact directly with the truncated form

of GAL4 suggesting that a single contact between a transactivator and a

component of the holoenzyme is sufficient to stimulate transcription (Barberis et

al. 1996; Farrell et al. 1996). Consistent with this idea, iûsion of a sequence-specific

DNA-binding protein to any single intrinsic component of Pol II holoenzyme,

such as GALll (Barberis et al. 1995; Farrell et al. 1996), FCPl (Archambault et al.

1996, submitted), or SR82 (Farrell et al. 1996), can trigger transcriptional activation

in vivo in the absence of a true transactivator. Furthermore, the SWI/SNF

cornplex associated with a Pol II holoenzyme complex isolated from yeast cells can

destabilize nucleosome structure in vitro (Wilson et al. 1996). These observations

suggest how transactivators might simultaneously remodel chromatin structure

and enhance the initiation of transcription in a gene-specific manner.

Although the holoenzyme mode1 provides a simplified view of the process

of transcriptional activation, the true physiological targets of transactivators

remain to be established. Indeed, in cases where transcription does not appear to

be limited by the rate of initiation, it is likely that the ability of transactivators to

interact with and modulate the activities of specific components of the Pol II

transcriptional machinery plays a crucial role in enhancing the rate of promoter

clearance and chain elongation by Pol II.

VIL Closing Comments

Although the studies highlighted above have provided broad insight into

the mechanisms by which transactivators function, several key issues remain

outstanding. First, we do not fully understand the biological function of many of

the accessory cornponents of the Pol II transcriptional machinery which appear to

regulate transcriptional activation in vizw. Second, we do not know the complete

range of specific protein-protein interactions mediated between transactivators

and components of the Pol II transcriptional machinery. Third, it remains unclear

how interactions between transactivators and one or more of the general

transcription factors trigger the molecular events associated with the initiation of

transcription, promoter clearance, and chah elongation by Pol II. Thus, it is

apparent that additional biochemical and genetic studies will be required for a

more complete understanding of the process of transcriptional activation.

VIII. Thesis Rationale

The identity of phvsiologically important targets of transactivators has

long been a controversial issue, in part because none of the studies reporting a

direct interaction between a transactivator and a component of the Pol II

transcriptional machinery (section V) were performed in the context of a

fûnctional promoter. Therefore, a long standing interest of mine has been to

devise a means of evaluating the protein-protein interactions mediated by a

transactivator under conditions which permit activation of transcription to occur.

1 have approached this issue by developing a systematic and controlled photo-

crosslinking assay.

The basis of my approach involved the selective chemical modification of

the activation domain of a transactivator with a photoreactive crosslinking

reagent. 1 first noted that the hetero-bifunctional crosslinking reagent maleimide-

4-benzophenone (MBP) could be selectively targeted to cysteine residues located

on the surface of a protein. MBP places a highly photoreactive benzophenone

substituent on the side-chain of cysteine residues which can then contact proteins

bound in relative proximity (< 10A) to the surface of the derivatized protein

(Chapter II). To exploit this observation, I created a novel recombinant

transactivator which had a single cysteine residue located on the surface of its

activation domain. 1 did this by expressing the acidic activation domain of the

human transactivator E2F-1, which encodes a single cysteine residue adjacent to

the binding surface for the retinoblastoma gene product, as a fusion to the

bacterial sequence-specific DNA-binding protein LexA. The LexA protein lacks

cysteine residues and has been used extensively to characterize heterologous

activation dornains (see, for example, Brent and Ptashne 1985; Ruden et al. 1991).

I then confirmed that this chimeric protein could activate Pol II-mediated

transcription in a sequence-dependent manner. As an important control, 1 also

carefully monitored that modification of the activation domain of LexA-E2F-1

with MBP did not impair its ability to activate transcription. The çuccess of the

crosslinking experirnents which are described in chapters II and III proved to be

dependent on the abundance and affinity of the targets of the transactivator, the

efficiency of crosslinking, and the sensitivity of detection.

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Prornoter-dependent Photo-Crosslinking of the Acidic Transcriptional

Activator E2F-1 to the TATA-Binding Protein

X version of this chapter was published in the Journal of Biological Chemistry Vol. 270,13674-13680,1995.

(1 did all of the experirnents in this chapter)

SUMMARY

Sequence-specific activators appear to increase the rate of initiatiot-t of

transcription by Pol II by contacting one or more of the Pol II general transcription

factors (reviewed in chapter 1). One candidate target of transactivators is the TATA-

binding protein, TBP, which nucleates the formation of a promoter preinitiation

complex subsequent to binding the TATA box. Using a site-directed pho toaffinity

crosslinking approach, 1 have shown that the acidic activation domain of the chimeric

activator LexA-E2F-I can interact with TBP when each of these factors is bound to a

transcriptionally responsive promoter. Mutations within the activation domain of

LexA-E2F-1 which impaired its ability to activate transcription in gitro were found to

reduce the binding of LexA-E2F-1 to promoter bound TBP. Although the association of

initiation factor TFIIB with the TBP-prornoter complex did not preclude the

interaction of LexA-E2F-1 with TBP, the regulatory factor TFIIA strongly inhibited

promoter-dependent crosslinking of LexA-E2F-1 to TBP. These results suggest that

acidic transactivators such as E2F-1 interact with TBP during the earliest stages in the

assembly of a preinitiation complex.

INTRODUCTION

RNA polymerase II (Pol II) requires a number of accessory protein factors in

order to initiate transcription accurately from a promoter. These general initiation

factors associate through extensive protein-protein interactions and recruit Pol II to

the promoter to f o m a preinitiation complex (reviewed in Ref. 1; Chapter 1).

Sequence-specifi c transcriptional activators stimulate transcriptional initiation by Pol

Il, at least in part, by facilitating the assembly of a productive preinitiation complex

(reviewed in Ref. 2). Indeed, transactivators might function at more than one step

during this process (3). Consistent with this possibility, the activation domains of

many transactivators have been shown to interact directly with one or more

components of the Pol II transcriptional machinery irt i~ i t ro . For example, the acidic

activation domain of herpes simplex protein VP16 has been reported to bind

independently to the TATA-binding subunit (Tf3P) of the general initiation factor

PI ID (4), to the general transcription factors TFIIT3 (5) and TFILH (6), as well as to the

transcriptional coactivators TAF40 (7) and PC4 (8) which associate with the

preinitiation complex. Similarly, the glutamine-rich activation domains of the

human transcription factor Spl have been shown to interact directly with both

TBP-associated factor TAFllO (9) as well as with TBP itself (10). The ability of

transactivators to interact with several different protein targets in the Po1 II

the

transcriptional rnachinery may account for the transcriptional synergy that is observed

with multiple promoter-bound transactivators. Nevertheless, given the multiplicity

of transactivator targets thus Çar proposed, the identity of biologically relevant target(s)

has remained sornewhat controversial. In this respect, it is noteworthy that most of

the studies reporting a direct interaction between a transactivator and a general

transcription factor have not been performed in the context of functional prornoter.

Therefore, it remains to be demonstrated if, and at what stage, such interactions occur

during preinitiation comp lex assembly or the initiation of transcription.

In a recent study of the process of transcriptional activation in prokaryotes,

photochernical crosslinking of proteins was used to characterize a promoter-

dependent interaction between the bacterial activator protein CAP and the CY subunit

of Escherichin coli RNA polymerase (11). 1 have developed a related strategy to study

protein-protein interactions mediated by a eukaryotic transactivator bound to a

cognate DDN eelment upstream of a Pol Il promoter. My approach involved the

selective derivatization of the acidic activation domain of a chimeric transactivator,

LexA-E2F-1, with a pho toreactive crosslinking rnoiety. Using this method, 1 showed

that LexAE2F-1 interacts directly with TBP when bound iipstream of a variety of Pol II

promoters. My results are consistent with the notion that TBP is an important target of

transactivators during the earliest stages in the assembly of a preinitiation complex

and establish the usefulness of the crosslinking approach to study the mechanisms of

transcriptional activation in eukaryotes.

EXPERIMENTAL PROCEDURES

Protein expression vectors

Bacterial expression vectors for LexA and LexA-E2F-1, each containing an N-

terminal polyhistidine tag, were prepared respectively by subcloning an

oligonucleotide linker encoding an translational stop codon and a DNA fragment

encoding amino acids 368-437 of E2F-1 (13) into the bacterial expression vector pJB07

(12). Truncated wild type (amino acids 400 to 437) and mutant E2F-1 activation domain

derivatives (13-17) were amplified by polymerase c h a h reaction and subcloned for

expression as LexA fusions into pJB07. The LexA constmcts were transformed for

expression into E. coli strains DH5a or JM107. DNA fragments encoding hl1 length

yeast TBP, human TBP, yeast TFIIB (Sua7), and human TFIIB were subcloned into the

bacterial expression vector pET19b (Novagen) or a pET19b derivative encoding the

recognition sequence for heart muscle kinase (18) adjacent to the N-terminal

polyhistidine tag and were transformed for expression into the E. coli strain

BL21(DE3).

Promoter cons tructs

An XbaI DNA fragment from pG5Lx2E4 (12) containing two LexA binding sites

was subcloned into the XbaI site upstream of the Ad2ML promoter in pAd2ML(A-50)

(19) and upstream of the CYCl promoter using an NheI site introduced in pGALCG-

(20). The same LexA binding sites were situated upstream of the HIS3 gene TR

promoter by subcloning a BamHl to Hindm DNA fragment from pG5Lx2E4 into

BamHl and HindIII digested pGCG17, pGC204, and pGC205 (21). The distance from the

proximal most LexA binding site to the (nearest) TATA element is 41, 36, and 41 base

pairs for the Ad2ML, CYCZ, and H1S3 T R promoter constructs respectively. Template

DNA was purified by cesium chloride density gradient centrifugation.

Protein expression and purification

Overnight cultures were diluted 1:15 into fresh LB media, grown at 30°C (for

PET-19 derivatives) or 37°C (for LexA derivatives) to 0D6m -1.0, and induced with

-300 pM isopropyl B-D-thiogalactopyranoside for 2-3 hrs. The bacterial pellets were

resuspended in buffer A (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HC1 pH 7.9, 5

mM B-mercaptoethanol) containing 1 mM phenylmethylsulfonyl fluoride and 5mM

benzamidine hydrochloride. The cells were sonicated on ice and the debris pelleted by

centrifugation (20,000g for 30 min at 4°C). Soluble extract was loaded at 8°C ont0 .++ columns containing Ni NTA agarose (400-500 pl bed volume; Qiagen) pre-

equilibrated with buffer A. The columns were washed successively with 5 coltimn

volumes each of buffer A, buffer A containing 45 mM imidazole, and buffer B (20 mM

HEPES-NaOH pH 7.9, 100 mM NaCl, 20% glycerol, 0.2 mM EDTA), and were eluted

with buffer B containing 1 mM DTT and 0.5 M imidazole (pH 7.9). Human TBP was

further purified by chromatography on heparin Sepharose (Pharmacia) as described

(10). The protein eluates were dialyzed extensively against buffer B containing 1 mM

DTT and stored at -70°C. Recombinant yeast TFIIA subunits TOAl and TOA2 were

each expressed in the strain BL21(DE3) and purified as recommended by the authors

(22). The TFIIA heterodimer was further purified by affinity chromatography on a

column containing immobilized recombinant yeast TBP coupIed to Affi-Gel 10 resin

(3mg/ml; BioRad). Bound TFIIA was eluted with buffer B containing 500 mM NaCl

and dialyzed extensively against buffer B containing 1 mM DTT.

Protein denvatization with the photo-crosslinker

10 pg of LexA-E2F-1 fusion protein were diluted with buffer C (20'' glycerol,

100 mM NaCI, 20 mM HEPES-KOH pH 7.0) containing 5 mM (3-merca~toethanol and

incubated with 50 pl (bed volume) ~ i + +NTA agarose beads (equilibrated with

buffer C) for 30 min at room temperature. The beads were then washed three times

with 1 ml of degassed buffer C . Under reduced lighting, maleimide-4-benzophenone

(Sigma) was then added frorn a freshly made 20 mM stock solution in dimethyl

formamide to a 10-fold molar excess relative to protein. After incubation in the dark

for 4 hrs a t room temperature, the beads were washed once with buffer C containing 5

mM B-mercaptoethanol. and the bound protein eluted witb. buffer B containing 1 mM

DTT and 0.5 M imidazole (pH 7.9). The solvent accessible thiol (ie cysteine) residues

were derivatized to >9O0L, as determined by titration with Ellman's reagent as

recommended by the manufacturer (Pierce). The photoreactive protein was stored

before use in amber microtubes at -70°C.

Protein radiolabeling

20 pg of TBP or TFIIB with N-terminal heart muscle kinase (HMK) tags were

treated with 20-40 units of HMK (catalytic subunit; Sigma) and 20-40 pCi of [ y 3 2 ~ ] ~ ~ ~

(6000 Ci/mmol)(NEN) in buffer B containing 1 mM DTT and 10 mM MgC12. After a 90

min incubation at 30°C, the mixture was loaded onto a NAP-5 gel filtration column

(Pharmacia) pre-equilibrated with buffer B containing 1 mM DTT. The column was

washed continuously with equilibration buffer and the excluded volume containing

the labeled protein collected. Control reactions using non-sequence tagged TBP and

TFIIB confirmed that the kinase labeled the recognition sequence tag site-specifically.

The labeled TBP was also found to support both basal and activated transcription

in vitro when added to a TBP-depleted yeast whole ce11 extract (data not shown).

Photo-crosslinking

Template DNA (approximately 0.5 pmol), 32~-labeled TEP or TFIIB and, where

appropriate, unlabeled transcription initiation factors were added to 35 pl of buffer D

(12 mM HEPESNaOH pH 7.9,60 mM K I , 12 '/O glycerol, 5 mM MgC12,l mM EDTA,

0.6 mM DTT) or yeast transcription buffer (Fig. 5)(23) contained in the wells of a

microtiter plate that had been pre-blocked ovemight with 10 mM Tris-HC1 pH 7.9, 100

M NaCl, 0.05% (v/v) Tween 20, 0.5% (w/v) gelatin. After incubation for 10 min at

23T, photoreactive Led-EZF-1 fusion protein was added under reduced light and the

incubation continued for an additional 30 min in the dark. The plates were then

placed on a UV-transilluminator (Fotodyne) and irradiated for 5 min to initiate

photolysis. Following irradiation, SDS-PAGE sample buffer was added and the

reaction mixtures transferred into microtubes and boiled. Crosslinked products were

separated by electrophoresis on 10% polyacrylamide gels containing SDS. The gels

were dried and exposed to film with a single intensifying screen for 12 to 24 hrs at

room temperature.

Immunoprecipitation and DNA mobility shift assay

For the immunoprecipitation analysis, 25 pl of a standard crosslinking reaction

were diluted with 500 pl of TTBS (0.05% Tween 20, 10 mM Tris-HC1 pH 7.9, 0.5 NaCl)

and incubated with rabbit antisera (2 pl) for 4 hrs on ice. Protein A-Sepharose beads (20

pl; Sigma) were then added and the incubation continued with rotation for 6 hrs at

8'C. The beads were subsequently washed five times with TTBS and boiled in SDS-

PAGE sample buffer. The bead supernatant was analyzed by electrophoresis on a 10%

polyacrylamide gel containing SDS followed by autoradiography. Addition of

ethidium bromide to 400 pg/ml in the incubation buffer to ensure a complete

inhibition of DNA binding by the proteins did not affect the precipitation efficiency

(data not shown). For the electrophoretic mobility shift assay, the proteins were

assembled in a 20 pl volume of crosslinking buffer (buffer D) and incubated for 20 min

at room temperature. The reactions were then run on a 5%-polyacrylamide native gel

(40:l rnono:bis ratio, 2.5% glycerol) in TGE buffer (25 mM Tris base, 190 mM glycine, 1

mM EDTA, final pH 8.5). Following electrophoresis for 2 hrs at room temperature, the

gel was dried and exposed to film.

Zn vitro transcription

In oitro transcription of the G-less cassette reporter templates was performed

essentially as described (23) with the following modifications. Reactions (30 pl)

contained 4.5 pl of yeast whole ce11 extract (80 mg/ml) prepared as described (25) from

the strain BI2168 (a prbl-1122, pep43, prcl-407), as well as 100 pM 3'-O-methyl GTP

(Pharmacia), 600 yM each of A T and UTP, 20 pM CTP, 20 units RNase Block 1

(Stratagene), 10 units RNaseTl (Boehringer), 2.5 pCi [ ~ ~ ~ P ] c T P (3000 Ci/mMol)(NEN-

Dupont), the appropriate template DNA (15-30 pg/ml), and carrier DNA (10 pg/rnl).

Reactions were assembled on ice and supplemented with recombinant transcription

factors as required. Transcription was initiated by the addition of NTPs and allowed to

proceed at 23'C for 45 min. The reactions were terminated by the addition of 10 u1 of

stop buffer (80 mM EDTA, 200 mM NaCl, 2% SDS) containing 100 pg of proteinase K

followed by incubation at 37'C for 20 min. The nucleic acids were precipitated with

carrier tRNA and isopropanol, boiled in deionized formamide, and separated on 6%-

polyacrylamide gels containhg urea. The gels were dried and exposed to film with a

single intensifying screen overnight at -70°C.

RESUCTS

Generation of the transactivator

To position a photoreactive crosslinking reagent uniquely within the activation

domain of a Pol II specific transcriptional activator, 1 generated a chimeric activator

consisting of the C-terminal acidic activation domain of the human transcription

factor E2F-1 (amino acids 368 to 437)(13, 15) fused to the bacteria1 sequence-specific

DNA-binding protein LexA (amino acids 1 to 202) (24). The resulting fusion protein

contained only a single cvsteine residue (Cys427 in E2F-1) which allowed for the site-

directed introduction of a thiol-reactive crosslinking reagent at a defined position

within the transactivator. 1 reasoned that the Cys427 residue would be exposed on the

surface of the E2F-1 activation domain and able to interact with the Pol II transcription

apparatus since it is located within the core of the E2F-1 activation domain and is

immediately adjacent to the binding site for the retinoblastoma gene product, a

repressor of activation by E2F-1 (15). As expected, purified recombinant LexA-E2F-1

(Fig. lA, Iane 2) was found to be a potent sequence-specific transcriptional activator of

the yeast CYCl promoter in in vitro reactions using a transcription competent yeast

whole ce11 extract (Fig. 1B; compare top and bottom panels). Activation was fully

attributed to the E2F-1 activation dornain since LexA alone did not stimulate

transcription in this system (Fig. 1B). The LexA-E2F-1 fusion protein was then

derivatized with the thiol-specific heterobifunctional photo-crosslinking reagent

maleimide-4-benzophenone, which has been used extensively to study protein-

protein interactions in vitro (25 and references therein). This permitted UV-induced

covalent crosslinking of LexA-E2F-1 to proteins that are in close proximity to the E2F-

1 activation domain (ie within a 10A radius from Cys427 of E2F-1; 25). Importantiy,

introduction of this crosslinking reagent did not impair the ability of LexA-E2F-1 to

activate in vitro transcription (Fig. 1C).

I I - 1 1

+ LexA sites

- LexA sites

LexA- - LexA E2F-1

C - MBP MOCK

Fig. 1. Purified transcription factors. A, purified recombinant proteins (2 pg of each) used in these studies. lane 1, LexA; lnne 2, LexA-E2F-1 (full-length activation domain); lmes 3 , 4 , mzd 5, kinase recognition sequence-tagged yeast TBP, human TBP and yeast TFW, respectively; lanes 6, 7,8, yeast T'FILA, hurnan TFIIB, and yeast TFW used in the

cornpetition assays. The proteins were run on a 12.5% polyacrylamide gel containing SDS and stained with Coomassie Blue. The sizes of the protein markers (M) are given to the right in kDa. B, sequence-specific transcriptional activation by the LexA-E2F-1 fusion protein. RNA transcripts (arrowheads) produced by in vitro transcription from the yeast CYCl promoter, with (upper panel) or without (lozuer panel) two upstream LexA-binding sites, in reactions containing yeast whole ce11 extract supplemented with either buffer alone (-), LexA or LexA-E2F-1 (5 pmol of eadi). Cf in vitro transcription driven kom the CYCl promoter in reactions supplemented with buffer alone or with

LexA-E2F-1 protein (5 pmol) that had either been derivatized with the crosslinker (MBP) or had been mock heated (MOCK).

Promoter-dependent crosslinking of LexA-E2F-1 to TBP

Since the activation domain of E2F-1 had been shown to interact with TBP in

solution (16; data not shown), 1 first assessed the ability of LexA-E2F-I to interact with

TBP when bound upstream of a Pol II promoter. To allow the detection of both free

and crosslinked TBP, the TBP used in these experiments was first radiolabeled with

3 2 ~ in vitro (see Experimental Procedures). CYCl promoter template DNA which was

responsive to LexA-E2F-1 in the in vitro transcription system was incubated with 3 2 ~ -

labeled yeast TBP and with photoreactive LexA-E2F-1 at a concentration (-170 nM)

similar to that used in the in vitro transcription analysis. The assembled ternarv

complexes were UV-irradiated to initiate photolysis and the resulting crosslinked

protein complexes subsequently detected by SDS-PAGE and autoradiography. As

shown in lane 2 of Fig. ZA, two closely spaced bands which had a mobility consistent

with the formation of a complex between LexA-E2F-I and TBP (ie 60-61 kDa) were

observed. Both of these complexes represented a covalent heterodimer of LexA-E2F-1

with TBP as they could each be specifically immunoprecipitated with antisera against

TBP (Fig. 2B; lanes 2 4 ) and LexA (lane 6) , but not with antisera against an u ~ e l a t e d

protein (lane 5). As the mobility of TBP on denaturing gels is particularly sensitive to

structural alteration (26), the formation of two activator-TBP complexes rnay be due to

the covalent attachment of LexA-E2F-1 to different residues on TBP. Importantly,

these same complexes were absent in control reactions that did not contain the

activator (Fig. 2A; lane 1) and were significantly reduced (2 6 fold) when a control

template which lacked LexA binding sites was used instead (lane 3). The residuai

crosslinking that occurred in the absence of the activator binding sites (lane 3) may

reflect a less stable interaction of the activator with TBP in solution (16). As described

below, 1 also found that LexA-E2F-1 could be crosslinked to human TBP in a similar

binding site-dependent marner, a result consistent with the evolutionary

crosslinked 1: complexes *

Fig. 2. Promoter-dependent crosslinking of an activator to TBP. A, SDS-PAGE

fractionation of UV-irradiated mixtures that contained 32~-labeled yeast TBP (3 pmol)

and, as indicated, photoreactive LexA-E2F-1 (7.5 pmol) and CYCl promoter DNA with

or without two upstream LexA-binding sites. The position of the crosslinked LexA-

E2F-1-TBP heterodirner complexes (bracket), free TBP (arrowhead), and an activator-

independent complex (*), possibly representing a TBP homodimer, produced in the

crosslinking reaction are indicated. B, crosslinking reactions, as in lnne 2 of A, were

precipitated with antisera specific to either yeast TBP, LexA, or B N 1 , an unrelated

RNA polymerase III transcription factor. Where shown, recombinant yeast TBP and

TFW (30 yg of each) were added as cornpetitor just prior to immunoprecipitation to

characterize the specificity of the TBP anti-sera.

conservation of the structure of TBP and the fact that E2F-1 is a transactivator of

human origin.

Specificity of covalent crosslinking of E2F-1 to TBP

The specificity of the photo-crosslinking reaction was evaluated in a series of

control experiments. As expected, crosslinking of the activator to TBP was found to be

highly contingent upon both UV-irradiation and derivatization of the activator with

the crosslinking reagent (Fig.3Af compare lanes 1 & 4 with lanes 2 & 3) since only a

Iow background level of crosslinking occurred in their absence. Crosslinking could be

specifically competed with an excess of unlabeled TBP (Fig. 38, lanes 2 & 3) but not

with the same quantity of the similarly charged protein lysozyme (lanes 4 & 5) ,

indicating that the interaction was specific. The C-terminal 37 amino residues of E2F-1,

which contain Cys 427, were sufficient for crosslinking to TBP (Fig. 3 8 , lane 5)

consistent with the observations that this same region of E2F-1 can function as an

activation domain both in mammalian cells in vivo (15) and in a yeast ce11 derived

extract iiz i~i tro (Fig. 3C, lane 2). In this context, mutation of the reactive cysteine

residue to alanine (C427A) reduced the level of crosslinking to TBP to a background

level (Fig. 3B, lane 6) although it did not noticeably affect transcriptional activity iiz

vitro (Fig. 3C, lane 3). Lnterestingly, crosslinking could be restored only partially by the

introduction of a cysteine residue at position 420 in E2F-1 (G420C) and very poorly

when introduced at position 411 (Y41lC)(Fig. 3A, lanes 6 & 7) although both mutants

strongly activated transcription in vitro (Fig. 3C, lanes 4 & 5). T'hese combined results

confirrn the specificity of the original crosslinking protocol and suggest that the

naturally occurring cysteine in E2F-1 lies near or within the activation domain surface

of E2F-1 that contacts TBP, although this residue is not essential pet- se for activation

domain function.

I I - L5

MBP + UV +

yTBP Lyzo -&a

Fig. 3. Site-specificity of the photo-crosslinking. A, SDS-PAGE analysis of cïosslinking

reactions that contained CYCl promoter DNA bearing two upstream LexA-binding

sites, 32~-labeled yeast TBP (3 pmol), and LexA-E2F-1 &ion proteins (7.5 pmol). LexA fusion protein containing a full-length (aa 368-437) E2F-1 activation domain was heated with the crosslinking reagent (MBP) and subjected to UV-irradiation as

indicated (lanes 1-4). The remaining reactions (lanes 5-5) contained, as indicated above

each lane, photoreactive LexA fusions to either a wild-type or mutant truncated (aa

400-437) E2F-1 activation domain and were al1 subjected to UV-irradiation.

B, crosslinking reactions performed as in Iane 1 of A, both in the absence (lane 1) or

presence of 30 or 60 pmol each of unlabeled TBP (Ianes 2 6 3) or lyzozyme control

protein (Innes 4 & 5) . Cf in vitro transcription of template DNA containing two LexA- binding sites upstream of the CYCl promoter in reactions supplemented with the LexA-E2F-1 derivatives shown in lnnes 5-8 of A (4 pmol of each).

Crosslinking requires a TATA element

To test the generality of the promoter-dependent interaction observed between

LexA-E2F-1 and TBP, 1 extended my analysis to the adenovims major late (AdZML)

promoter. The Ad2ML promoter has been used extensively in in uitro studies of

transcription and contains a single TATA-element, unlike the CYCl promoter which

has three distinct TATA elements. As with the CYCl promoter constructs, purified

LexA-E2F-1 could both strongly activate in uitro transcription (Fig. 4A. compare top

and bottom panels) and be crosslinked to TBP (Fig. 4B) in a sequence-dependent

manner at the Ad2ML promoter. Quantitation of the data shown in Fig. 48 indicated

that 0.15 pmol of TEP was crosslinked to the activator in the presence of 0.5 pmol of

AdZML promoter DNA. Since the majority of the crosslinked complexes formed in a

promoter-dependent manner, I infer that a productive (ie crosslinkable) interaction

occurred between the activator and TBP at nearly 30% of the available promoters.

To confirm that the TBP that had been crosslinked to the activator had itself been in

contact with the promoter (ie bound to the TATA element), 1 compared the ability of

LexA-E2F-1 to interact with TBP at a promoter containing either a wild type TATA

eIement (derived from the H1S3 gene T R promoter; 21) or one of two mutant TATA

elements previously shown to be defective for binding to TBP (21; 27). As seen in Fig.

4D, crosslinking of Lefi-E2F-1 to TBP was largely restricted to the promoter bearing a

functional wild type TATA-element. Of the three templates studied, only this same

construct was transcriptionally responsive to LexA-EZF-l in vitro (Fig. 4C). Therefore,

a physical and functional interaction between LexA-E2F-1 and TBP occurred

preferentially when each of these factors was bound to their respective promoter

elements.

LexA- LexA- - LexA E2F-1 - LexA E2F-1

+ LexA sites

- LexA sites

B D < K U

LexA a z $ sites + 5 p p

Fig. 4. Interaction of the activator with TBP at other promoters. A, RNA transcripts

produced by in vitro transcription of Ad2ML promoter template DNA with (tipprr pnnel) or without (lower pnnel) two upstream LexA-binding sites in reactions

supplemented with buffer alone (-), LexA or full-length LexA-E2F-1 (5 prnol of each).

8, SDS-PAGE fractionation of UV-irradiated mixtures that contained photoreactive

full-length LexA-E2F-1 (7.5 pmol), 32Plabeled yeast TBP (3 prnol), and Ad2ML

promoter DNA with (lnne 1) or without (lane 2) two upstream LexA-binding sites. C, transcriptional activation of the yeast HIS3 gene TR promoter bearing a wild type

(TATAA) or a mutant (TGTAA, TCTAA) TATA element. D, SDS-PAGE fractionation of UV-irradiated mixtures that contained 32~-labeled yeast TBP (3 pmol),

photoreactive LexA-E2F-1 (7.5 pmol), and DNA bearing two LexA-binding sites upstream of the HIS3 TR promoter that had either a wild-type or mutant TATA

element.

Crosslinking correlates with transactivation

To ascertain the functional relevance of this promoter-dependent interaction

between LexA-E2F-1 and yeast TBP, I analyzed the effects of a series of mutations in the

E2F-1 activation domain on both in oitro transcriptional activation and crosslinking

by LexA-E2F-1. A total of ten mutant derivatives were expressed and purified as

fusions to LexA (Fig. 5A, top panel). Ail of the fusion proteins bound DNA with a

similar efficiency relative to the wild type LexA-E2F-1 fusion (data not shown). 1 first

established the relative strengths of the wild type and mutant LexA-E2F-1 derivatives

to activate iiz i~i tro transcription from the CYCl reporter gene. Consistent with the

results of transcriptional studies performed in vioo (14-16), the different E2F-1

activation domain mutants exhibited a range of transcriptional activity in vitro

relative to the wild type construct (Fig. 5A, rniddle panel). The proteins were each then

derivatized with the crosslinking reagent and assessed for their ability to interact with

TBP at the same promoter (Fig. 5A, bottom panel). To a large extent, the degree of

promoter-dependent crosslinking to TBP achieved with each of the LexAE2F-1

deriva tives correlated d irectly with their respective ab ili ties to activa te transcription

in vitro (for a quantitative cornparison, see Fig. 58). For example, the mutations which

exhibited the greatest reduction in crosslinking to TBP (e-g. L4lSP/ LKXP,

Y411A/F413A) were the same mutations that most dramatically impaired

transcriptional activation. On the other hand, mutants which displayed a more

modest reduction in crosslinking to TBP (e.g. F429P, de1420-422/Y411A) had a

correspondingly less pronounced effect on transactivation. While, in general, the

mutations had a more pronounced effect on transcriptional activation in uitro as

compared to their effect on transcriptional activation in vivo (14-16) and on

crosslinking to yeast TE3P, taken on the whole, the results of this analysis support the

notion that the promoter-dependent interaction of LexA-E2F-1 with TBP plays an

important role in the transactivation process. One mutant (T433A/P434A) that was

rcj P, V, r i -

% Activity

impaired in its ability to activate transcription in vitro did not, howwer, display a

noticeably reduced ability to be crossluiked to TBP. This suggests that the activation

domain of E2F-1, like the activation domain of VP16, may also interact with additional

cornponents of the Pol II transcriptional apparatus such as TFW (5), TFIH (6) , or TAFs

during the transactivation process.

Effects of TFIIA and TFIIB on crosslinking

The transcription stimulatory factor TFIIA and the general initiation factor

TFW can each interact independently with TBP at a promoter (reviewed in Ref. 1). To

investigate whether LexA-E2F-1 could still bind to TBP that waç present in such early

intermediates of preinitiation complex assembly, 1 performed the crosslinking reaction

in the presence of purified TFIIA or TFIIB. The recombinant TRIA and TFIIB (Fig. IA)

readily formed characteristic complexes with TBP that could be visualized uçing an

electrophoretic mobility shift assay (Fig. 6A). Surprisingly, preincubation of yeast TBP

with an equimolar amount of yeast TFIIA resulted in a nearly complete inhibition of

crosslinking of LexA-E2F-1 to TBP whether using the yeast CYCl promoter (Fig. 6B) or

the mammalian Ad2ML promoter (Fig. 6C) as template. 1 also found that yeast TFIIA

could inhibit crosslinking of LexA-E2F-I to human TBP (Fig. 6D), a result consistent

with the ability of yeast TFIIA to bind human TBP (28). In contrast, preincubation of

yeast TFIIB with yeast TBP (Fig. 68, C) or hurnan TFITB with human TBP (Fig. 6D)

resulted in each case in only a modest reduction in the level of crosslinking of LexA-

E2F-1 to the Tl3P target. These results suggest that the activation domain of E2F-1 can

bind to TBP that is complexed with TFIIB but cannot do so in the presence of TFIIA.

Since TFIIB has itself been reported to bind directly with acidic transcriptional

transactivators (5), 1 performed an analogous serieç of crosslinking experiments using

32~4abeled TFW, as well as TBP, as the target. As shown in Fig. 7A. LexA-E2F-1

y T B p - + + + + + + + yllA yTFIIB - - + - - + - + yilA + yIIB + yllB

huTFIlB - - - + - - + LexA I -7

yTFIIA - - - + + + sites + - + - + + -

C +yiIA +y116 D + yllA +huIlB LexA I- LexA n- sites + - + - + - sites + - + - + -

Fig. 6. Effects of TF?IA and TFIIB on the activator-TB1 interaction. A, non-denaturing SDS gel showing binding of yeast TBP (25 ng) to 32~-labeled Ad2ML promoter DNA

both in the absence or presence of yeast TFIIA, yeast TFW, and human TFW (50 ng of each). DNA-bound complexes containing TBP (D), TFW (B), and/or T F W (A) are

indicated to the right. B, SDS-PAGE fractionation of UV-irradiated mixtures that

contained %'-labeled yeast TBP (3 prnol), photoreactive full-length LexA-EZF-1 (7.5

prnol), CYCl promoter DNA with or without two LexA binding sites, and yeast TFIW ( y u ) or TFW (yW) (3 pmol of each) as indicated. C, SDS-PAGE fractionation of UV- irradiated mixtures that contained 32~-labeled yeast TBP (3 pmol), photoreactive full- length LexA-EZF-1 (7.5 pmol), and Ad2ML promoter DNA with or without two

upstream Led-binding sites. The reactions were supplemented with yeast TFIW and yeast TFW (3 pmol of eadi) as indicated. D, SDSPAGE fractionation of UV-irradiated mixtures that contained 32~-labeled human TBP (3 prnol), photoreactive full-length LexA-E2F-1 (7.5 pmol), CYCl promoter DNA with or without two upstream L e u - binding sites, and either yeast TFIIA or human TFIIB (hum) (3 pmol of each).

could be crosslinked to yeast TFIIB (compare lanes 4 & 7 to lane 3) alrnost as efficiently

as to yeast TBP (lanes 2 & 5 ) . This interaction, like that of LexA-E2F-I with TBP,

appeared to be specific in that it could be competed with an excess of unlabeled TFW

(Fig. 7l3, lanes 2 and 3) or TBP (lanes 6 and 7) but not with a similar amount of the

control protein lysozyme (lanes 4 and 5). While these results are consistent with the

notion that TFW might also be a target of the activation domain of E2F-1, an

essentially identical level of crosslinking of L e A - E X - 1 to TFIIB occurred both in the

presence (Fig. 7Ar lanes 4 & 5 ) or absence of TBP (lanes 6 & 7) or activator-bindhg sites

in the template DNA (lanes 6 & 4). Therefore, unlike its interaction with TBP, the

interaction of LexA-E2F-1 with TFIIB did not exhibit any promoter-dependency.

LexA-E2F-1 + + - + + + + yTFIIB- - + + + + + y T B P + + - + + - -

LexA sites - + + - + - + . . . , .. - - 97

yllB Lyzo yTBP - A & A

Fig. 7. Crosslinking of the activator with TFIIB. A, SDS-PAGE fractionation of UV-

irradiated mixtures that contained, as indicated, 3%'-labeled yeaçt TFUB and/or yeas t

TBP (3 pmol of each), photoreactive full-length Led-E2F-1 (7.5 pmol), and CYCl

promoter DNA with or without upstream LexA-binding sites. Crosslinked complexes

containing LexA-E2F-1 covalently attached to either T'Fm or TBP are indicated by the

arrow and bracket respectively. B, crosslinking of LexA-E2F-1 to TFW in the absence

(lane 1) or presence of 30 or 60 pmol of unlabeled TFW (lnnes 2 6 3), yeast TaP (lnnes 6

8 7), or lysozyme (lanes 4 G. 5).

DISCUSSION

In this chapter, 1 provide direct biochemical evidence that a hanscriptional

activator can interact with TBP when bomd upstrearn of a Pol II promoter. Although

largely consistent with previous reports that have implicated TBP as a target for

transcriptional transactivators, my study suggests that this interaction occurs

preferentially once TBP is itself bound to the promoter. The observation that

transactivators can, under certain conditions, interact with TBP in the absence of DNA

(4, 10, 16) may be due to the use of a significantly higher concentration of activator

protein in those studies, which relied on affinity chromatographic techniques. In the

photochemical crosslinking approach described here, I have used a markedly lower

concentration of activator that is, nevertheless, sufficient for transcriptional activation

in oitro. The interaction between activators and TBP may be stabilized by mutual

interaction with DNA. Two lines of evidence, however, suggest that the ability of an

activator to interact with promoter bound TBP is biologically relevant. First,

LexA-E2F-I was found to interact preferentially with TBP at three distinct Pol II

promoters that were responsive to this activator. Second, 1 found that mutations that

reduced the ability of LexA-E2F-1 to crosslink with T6P at the CYCl promoter

concomitantly affected the ability of the activator to stimulate transcription in oitro

from this same promoter.

Although the interaction of an activator with TBP may facilitate the binding of

TBP to a promoter in vivo (29), my results suggest that TBP remains an important

target of transactivators even after it has been recruited to a promoter. It is possible

that by directly contacting TBP at a promoter, an activator like LexA-E2F-1 or GAL4-

VP16 may displace inhibitors of transcription associated with TBP à1 iiivo which

impede the formation a productive preinitiation complex (31). Alternatively, the

activator might confer a conformational change in the promoter-bound TBP in a

manner that facilitates the subsequent recruitment of other general initiation factors,

such as TFIIB, to the promoter (2,30,32). Consistent with this latter possibility, specific

point mutations in TBP which show a defective transcriptional response to GAL4-

W16 hinder GAL4-VPl6 mediated recruitment of TFIIB to the initiation complex (30).

Although the recruitment of TFIIB to the promoter can be a rate-limiting step in the

initiation of transcription, it need not be the only step accelerated by transactivators.

For example, the ability of LexA-E2F-1 (this study) and GAL4VP16 (5) to interact

directly with TFm may, in tum, facilitate the association of Pol II and TFIIF with the

preinitia tion complex.

TFIIA is required for efficient transactivator function under certain conditions

in vitro (28,33-35) and the formation of a preinitiation complex containing TFIIA is

thought to be an important step in the transactivation process (36). In aggreement with

observations by Liljelund et nl. (37), we found that TFIIA inhibits the binding of an

acidic activator (ie LexA-E2F-1) with TBP at both yeast and mammalian promoters.

This result suggests that the acidic activation domain of E2F-1 binds to an overlapping

region or surface of TBP that is also contacted by TFTIA. TFIIA may also alter the

conformation of the TBP-promoter complex in such a way as to preclude the

subsequent association of an activator with TBP. Alternatively, a more trivial

explanation is that TFIIA intefers with the ability of the crosslinker to contact the

surface of TBP. This is likely given that the interaction of transactivators with TBP is

thought to assist in the recruitment of TFIIA to the promoter by dispiacing inhibitors

of transcription which bind to TBP and block the association of TFIIA with the TBP-

promoter complex (31). Although I used yeast TFIIA in this study, 1 expect that human

TFIIA will also display a similar ability to inhibit the crosslinking of E2F-1 to TBP since

both homologs are structurally and functionally conserved (28). In contrast, neither

yeast TFIIB or hurnan TFIIB appears to block the crosslinking of LexA-E2F-1 to TBP.

Thus, TBP can be a target of an activator even after the association of TFIIB with the

preinitiation complex. Following the association of TFIIA with the TBP-promoter

complex, the activator may become displaced from its contact with TBP and would

then be free to interact with other components of the transcription apparatus,

including those that function at a later stage in the initiation process such as TFIM (6).

Crosslinking experiments similar to those reported here but performed in the context

of a complete activator responsive system may help to resolve the range of

interactions mediated by a transcriptional activator with the Pol II transcriptional

machinery.

I thank D. Cress, C. Hagemeier, E. Harlow, W. Kaelin, and T. Kouzarides for

generously providing E2F-1 cDNA derivatives. We also thank J. Brickman and M.

Ptashne for the LexA expression vector, R. Brent for antibody to L e d , and R. Ebright

and J. Greenblatt for helpful advice.

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c m III

Identification of a Novel Target of Transcriptional Activators

by Photo-Crosslinking.

A version of this chapter will be submitted as a manuscript for publication.

(The protein rnicrosequencing in this chapter was done by Ryuji Kobayashi [CSHL])

I I I - 1

SUMMARY

As described in chapter II, 1 have developed a sensitive and highly specific in

vitro crosslinking strategy to identify protein targets of the chimeric activator LexA-

E2F-1. Here, 1 report that the activation domain of LexA-E2F-1 interacts in a promoter-

dependent manner with a novel component of the yeast pol II transcriptional

maciinery, XTC1. 1 also show that XTCl interacts directly with the activation domains

of the herpes virion protein VP16 and the yeast activator GAL4 suggesting it is a

comrnon target of activators. Finally, 1 found that yeast strains deleted for the XTCl

gene exhibit growth defects and altered responses of the pol II transcriptional

machinery to activators in vivo, consistent with XTCl being a physiologically

relevant target of activators in yeast.

INTRODUCTION

Eukaryotes employ multiple mechanisms to ensure that cellular synthesis of

mRNA is tailored to changing environmental and developmental cues and

physiological requirements. The activity of Pol II is regulated through the combined

action of gene-specific transcription factors which bind to cognate sequences upstream of

a promoter and positively- and negatively-acting transcriptional cofactors which are

intrinsic components of the Pol II transcriptional machinery (reviewed in chapter 1; refs.

1 and 2). Transactivators, such as the human proto-oncoprotein E2F-1 or the yeast

protein GAL4 appear to stimulate initiation, promoter clearance, and/or chah

elongation by pol II by interacting directly with one or more of components of the Pol II

transcriptional machinery (reviewed by refs. 3-5).

In one model of the activation process, a promoter bound transactivator

stimulates transcription by interacting with distinct components of the Pol II

transcriptional machinery in multiple discrete steps. One prediction of this model is that

each interaction mediated by a transactivator is likely to be crucial for the subsequent

formation of a productive preinitiation complex at a promoter. However, the recent

characterization of large protein complexes containing Pol II and most of the general

transcription factors (chapter 1) suggests that transactivators recruit the entire Pol II

transcriptional machinery to a promoter in a single step. Consistent with this notion, it

has been observed that artificial recruitment of the Pol II holoenzyme to a promoter

results in activated levels of transcription in the absence of a transactivator (chapter 1).

This alternative holoenzyme pathway for transcriptional activation suggests that there

may be a degree of functional redundancy in the nature of the interactions mediated

between an activator and the Pol II transcription machinery needed to influence

transcription. Elucidation of the functional targets of transactivators is therefore

essential to understanding the molecular details of the control of gene expression.

To better understand the mechanisms involved in the activation of Pol II

mediated transcription, 1 sought to identify the direct protein targets of a transactivator

using a highly selective iri zdro crosslinking strategy. As described in chapter II, the basis

of this approach involved the positioning of a photoreactive crosslinking moiety within

the activation domain of the transactivator LexA-EZF-1. Using this method, 1 found a

direct interaction between the activation domain of LexAE2F-1 and a novel component

of the Pol II transcrip tional machinery, XTC1. The XTC1 gene product has characteris tics

of a negative regulator of transcription and a target of the activation process. My results

suggest that transactivators function, at least in part, by relieving repression of

transcription by Pol II.

Plasmid constructs. LexA-E2F-1 was cloned into a modified pETl9b E. coli expression

vector (Novagen) encoding tandem N-terminal poly(l0)histidine and heart muscle

kinase tags6. The XTCl ORF was amplified from genomic DNA and subcloned for

expression into pET19b. The GST constructs and promoter DNA ternplates are described

e l~ewhere~? '~ ; the H1S3 TR TATA box was deleted by digestion at flanking restriction sites

followed by religation. Protein expression and purification was as described6? The

overexpression vector for GAL4-E2F-1 (amino acids 387-437 of E2F-1 fused to the DNA-

binding domain of CAL$) was kindly provided by A. Pearson and J. Greenblatt.

Extract preparation. Whole ce11 extracts from the strain DPY213 (ref. 27) or an isogenic

x t c l A strain were prepared and fractionated by chromatography on a Bio-Rex70 column

essentially as described2'. The 0.6 M K acetate fraction was used in the crosslinking

experiments after extensive dialysis against transcription buffer (50 mM HEPES-KOH, 90

mM K acetate, 10% glycerol, 10 mM Mg acetate, 2 mM EGTA, and 2 mM DTT, pH 7.6).

The final protein concentration of the extract was -10 mg/ml.

br vitro transcription and photo-crosslinking. Recombinant LexA-E2F-1 was labelled to

high specific activity (-2x105 cpm/pmol) with [$2P]~TP (6000 Ci/mmol; NEN) using

heart muscle kinase essentially as described6. The labelled protein was bound to Ni-NTA

agarose beads (Qiagen), washed extensively with buffer C (ref. 6), and derivatized with

maleimide-4-benzophenone (Sigma) as described6. The beads were then washed with

buffer C and eluted with transcription buffer containing 0.5 M imidazole. Photoreactive

LexA-E2F-I (3 pmol) was mixed with yeast extract (0.2 mg protein), template DNA (0.5

pmol), recombinant yeast TFIIA (3 prnol)6, and transcription buffer in a total volume of

25 pl. The mixtures were irradiated for 12 min under a UV-transilluminator (UVP

mode1 TM-36; -8500 w / cm2/sec) and fractionated on a 7.5%-polyacrylamide gel. The

gels were dried and expoçed to film for 12 hrs at -70°C. RNA synthesis was measured in

the absence of UV-irradiation as described6.

Affinity chromatography. Proteins were coupled to AffiGel 10 resin (Bio-Rad) to a final

concentration of 2 mg/ml. Micro-colurnns were loaded with either 0.4 mg of yeast extract

or 2 pg of recombinant XTC1, washed with 10 volumes of transcription buffer, and eluted

with buffer containing 1M NaCl. For microsequencing, 40 ml of the yeast transcription

extract were loaded ont0 a LexA-E2F-1 affinity column (2 ml). The bound proteins were

eluted with 1M NaCl, concentrated, and fractionated on a 12.5°/~-polyacry1amide gel.

Protein sequencing was as described2' and was perforrned by R. Kobayashi (CSHL).

Immunoprecipitation. Antibodies were raised by immunizing rabbih with recombinant

XTC1. For imrnunoprecipitation, 20 pl of a standard crosslinking reaction was diluted

with 500 pl of TTBS (0.05% Tween 20, 10 mM Tris-HC1 pH 7.9, 0.5 NaCl) and incubated

with rabbit antisera (2 pl) for 4 hrs on ice. Protein A-Sepharose beads were then added

and the incubation continued with rotation for 6 hrs at 4°C. The beads were washed

extensively with TTBS and boiled in sample buffer.

Yeaçt Growth and Manipulation. Cells were transformed by the lithium acetate

technique3' and grown in YPD or minimal medium supplemented with appropriate

nutrients. The XTCl ORF in the yeast diploid strain LP112 (ref. 22), the haploid strain

DPY213 (ref. 27), or the haploid strain YCJ0032 (ref. 22) was replaced with a LEU2 or TRPI

gene cassette by a standard replacement procedure. Gene dismption was verified by PCR.

Analysis of fi-galactosidase activities was performed as described2' and normalized to the

0D595 of the cultures and the assay time.

RESULTS

To study interactions between

AND DISCUSSION

a n activator and components of the pol II

transcriptional rnachinery, I performed in vitro crosslinking experiments using a

radiolabelled, photoreactive derivative of the chimeric activator LexA-E2F-1 (ref. 6).

This chimera consists of the acidic C-terminal activation domain of the human

activator E2F-1 (amino acids 400 to 437; ref. 7) fused to the bacterial DNA-binding

protein LexA and is a potent activator of transcription when bound upstream of a

pol II prornoter both in yeast ce11 extracts6 and in yeast cells (Table 1, line 7). 1 labelled

purified recombinant LexA-E2F-1 with 3 2 ~ and then derivatized it with the hetero-

bifunctional crosslinking reagent maleimide-4-benzophenone (MBP)~.' at the single

cysteine residue located within its activation domain, residue 427 in E2F-1 (Fig. In) .

The derivatized activation domain is capable of interacting with the pol II

transcrip tional

transcription in

machinery (Fig. -

machinery as MBP-derivatized LexA-E2F-1

a yeast extract enriched for components of the

strongly activated

pol 11 transcriptional

'1 6, see also ref. 6).

'1.0 capture protein interactions mediated by LexA-E2F-1 during the process of

transcriptional activation, 1 UV-irradiated yeast transcription extract following the

addition of 32~-labelled, MBP-derivatized LexA-E2F-1 and a responsive promoter DNA

template. As seen in lane 1 of the autoradiogram shown in Fig. lc, several distinct

crosslinked complexes consisting of LexA-E2F-1 covalently bound to proteins in the

extract were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Crosslinking of LexA-E2F-1 to these proteins did not occur in the absence of UV-

irradiation (lane 2) or pre-treatrnent of the activator with MBP (lane 3). Mutation of the

reactive cysteine residue in LexA-E2F-1 to alanine also eliminated crosslinking (lane 4)

confirming that the crosslinking was mediated through the derivatized cysteine residue

CYCl HIS3

Figure 1. Selective crossIinking of an activator to proteins in a yeast extract. a, The 32P-labelled, MBP-derîvatized LexA-EX-1 fusion protein. b, Autoradiogram of RNA transcripts produced by a yeast extract with (lane 2) or without (lane 1) addition of MBP-LexA-E2F-1. The DNA template contained two LexA-binding sites upstream of the CYCl prornoter.~, Autoradiogram of SDSPAGE fractionated crosslinking reactions containing MBP-LexA-E2F-1, yeast extract, and the DNA template used in b. All reactions except for lane 2 were UV-irradiated. In lane 3, LexA-E2F-1 was not pretreated with MBP. Crosslinking by LexA-E2F-1 denvatives without a cysteine or with a cysteine at amino acid 411 or 420 in E2F-1 is shown in lanes 4,5, and 6, respectively. Mr of protein markers is given in kDa; 32P-labelled LexA-E2F-1, *. d, Crosslinking by LexA- E2F-1 in the presence of DNA templates with (+) or without (0 ) LexA-binding sites or a TATA element. The templates contained either the CYCl (lanes 1 and 2) or the HIS3 TR (lanes 3 and 4) promoters. Promoter-dependent complexes 1 - III are indicated. e, Crosslinking by LexA fusions to a wild type (Iane 1) or a mutant, D428A/F429Af derivative (lane 2) of the E2F-1 activation domain. The DNA template was as in b.

within the E2F-1 activation domain. Furthermore, this crosslinking by LexA-E2F-1

was selective since the pattern of crosslinked complexes waç altered by positioning the

reactive cysteine residue, and therefore the crosslinker, at either of two different

positions within the E2F-1 activation domain (lanes 5 and 6) in a manner which does

not, however, impair the ability of LexA-E2F-1 to activate transcription6.

To provide evidence that one or more of these crosslinked complexes was the

result of an interaction between LexA-E2F-1 and a bone fide component of the yeast

pol II transcriptional machinery, 1 performed identical crosslinking reactions using

DNA templates with or without LexA-binding sites or a TATA element. As seen in

Fig. Id , three of the more prominent crosslinked complexes (1, II, and III) formed

preferentially when LexA-binding sites were present upstream of a promoter

(compare lanes 1 and 2). Formation of these same complexes was markedly reduced,

however, when the DNA template lacked a TATA element (compare lanes 3 and 4).

Furthermore, the formation of these complexes appeared to be closely linked to the

activation process since their appearance was reduced by a double point mutation

(D428A/F429A) in the E2F-1 activation domain (Fig. le ) which impairs the ability of

LexA-E2F-1 to activate transcription (Table 1, compare lines 7 and 8; ref. 6). The

selectivity of this crosslinking was therefore consistent with the type of interactions

expected of an activator.

To identify proteins which crosslinked to LexA-E2F-1, 1 first fractionated the yeast

extract by chromatography over an affinity column containing immobilized LexA-E2F-1.

The bound proteins were eluted with a high salt buffer and were either visualized by

silver staining of an analytical SDS gel (Fig. 2n) or were dialyzed against transcription

buffer and UV-irradiated in the presence of MBP-LexkE2F-1. As seen in Fig. 2b,

complexes 1-111 again formed specifically between LexA-E2F-1 and proteins eluted from

the LexkE2F-1 affinity column (lane 2) but not with the eluate of a LexA control

I I I - 1 O

Figure 2. Purification and cloning of XTC1. LI, Silver stained SDS gel of eluates from

LexA-E2F-1 (Iane 1) and LexA (lane 2) affinity columns. The 28 kDa LexA-E2F-1- binding protein is indicated by an arrow. 6, Autoradiograph of crosslinking by MBP-

LexA-E2F-1 in a yeast extract (lane l), in eluates from LexA-E2F-1 and LexA affinity

columns (lanes 2 and 3), or in the absence of added protein (lane 4). c, Amino acid

sequence of XTC1; residues obtained by rnicrosequence analysis are underlined.

ci , Sequence-alignment of XTCl (middle) with yeast (top) and human (bottom)

RAD54; sequence identities and conserved residues are indicated by bars and colons,

respective1y.e. Autoradiograph of crosslinking by MBP-LexA-E2F-1 in extracts from

isogenic wild type (lane 1) and XTCl deficient (lane 2) yeast; preimmune serum (Iane

3) or anti-XTC1 serum (lane 4) immunoprecipitates of a wild type yeast extract after

UV-irradiation of MBP-LexA-E2F-1; crosslinking by MBP-LexA-E2F-1 in the absence

(lane 5) or presence (lane 6) of recombinant XTCl (200ng).

I I I - 1 1

column (lane 3) or in the absence of added extract (lane 4), indicating that the

crosslinking targets of LexA-E2F-1 interacted specifically with the LexA-E2F-1 ligand.

Since the 28 kDa protein in the LexA-E2F-I column eluate was consistent with

it being responsible for the formation of complex I (-60 kDa) in the crosslinking

reactions with MBP-LexA-E2F-1 (-34 kDa), 1 scaled up the purification procedure to

permit direct microsequencing of this protein. A 17-mer peptide sequence,

LIQRVGNIAREESVILK, was obtained and found to match perfectly to a portion of an

open reading frame (D9740.9) located on chromosome IV of S. cc.revisine which

encodes a previously uncharacterized protein of 226 amino acids in length (calculated

M r 26,895, pI 9.53) with no obvious structural motifs (Fig. 2c) . This protein, which I

have narned XTCl (for Crosslinked lranscription Çomponent l), exhibits significant

sequence similarity (residues 75 to 216 in XTCl) to yeast and human RAD54 in a

region encompassing their canonical helicase motifs IV, V, and VI (ref. 9; GenBank

accession number X97795; Fig. 2d). The much smaller XTCl protein, however, lacks

motifs Ia, Ib, II, and III needed for helicase/ ATPase activity 1°.

To confirm that complex 1 consisted of XTCl crosslinked to LexA-EZF-l, I first

showed that extract prepared from yeast cells deleted for the XTCl gene did not form

complex 1 in crosslinking reactions with MBFLexA-E2F-1 (Fig. 2e, compare lanes 1 and

2) although formation of the other crosslinked complexes was largely unaffected.

Second, 1 found that complex 1 could be selectively irnmuno-precipitated using

antibodies raised against recombinant XTCl (lane 4) but not with preimmune serum

(lane 3). Third, 1 found that recombinant XTCl formed a crosslinked complex with MBP-

LexA-E2F-1 of the same mobility on SDS-PAGE as complex 1 (lane 6). Finally, in affinity

chromatography experiments, 1 found that both XTCl present in yeast extract and

recombinant XTCl bound well to immobilized LexA-E2F-1 (Fig. 3, lane 3). In contrast,

XTCI bound poorly to the mutant derivative of LexA-E2F-1 (lane 4) that was

Figure 3. XTCl interacts with the activation domains of several activators.

(Upper panel) hmunoblot probed with anti-XTC1 of an SDS gel showing fractionated

input yeast extract (lane 1) and high salt eluates (lanes 2-9) from different affinity

columns. The immobilized ligand is indicated above each lane. (Lower panel)

Coomassie Blue staining of an SDS gel showing input recombinant XTCl (lane 1) and

the high salt eluates (lanes 2-9) from a similar set of affinity colurnns. For both panels,

20% of the input and 50% of the column eluates were run on the gel. XTCl is indicated

by an arrow.

impaired for transcriptional activation (Table 1, line 8) and formation of complex 1

(Fig. le).

XTCl appears to be a target of a number of activators in addition to LexPL-EX-1.

In affinity chromatography experiments, XTCl bound well to GST-fusions encoding

the C-terminal activation domains of either the yeast activator GAM (amino acids

841-874; ref. 11) or the herpes virion protein W16 (amino acids 413-490; ref. 12) (Fig. 3,

lanes 5 and 7). Truncation of the activation domain of VP16 a t amino acid 456 or

mutation of the critical phenylalanine 442 to proline, changes known to affect the

activation potential of VP16 (ref. 13), reduced or eliminated binding of XTCl to VP16

respectively (lanes 8 and 9). Therefore, the ability of activators to interact directly with

XTCl correlated with their ability to activate transcription.

Consistent with the crosslinking data, XTCl copurified with pol II and its

associated general transcription factors upon fractionation of a yeast extract on BioRex 70

and DEAE-Sepharose ion-exchange columns (data not shown) and was present in a

preparation of yeast pol 11 holoenzyme isolated on a column containing immobilized

TFIIS (Fig. 40; G. Pan, T. Aso, & J. Greenblatt, manuscript submitted). Like loss-of-

function mutations in other accessory components of the pol II holoenzyme, such as the

SRB, ADA, and SWI/SNF transcriptional cofa~tors '~ - l~ , yeast strains deleted for the

XTCl gene (xtcld) grew slowly and were temperature sensitive for growth on synthetic

media containing glucose (Fig. 4b) and were unable to grow using galactose as the sole

carbon source (Fig. 44. This latter defect was a result of XTCl deficiency since galactose

prototrophy was restored by ectopic expression of XTCl (Fig. 4c).

To determine if the growth defects exhibited by an xtc1A strain were associated

with an impaired response to activators, 1 compared the ability of xfc1A and isogenic

wild type strains to support activated levels of transcription (Table 1). Transcriptional

activation by endogenous GAL4 was monitored u s h g both a single copy integrated

GAL4 responsive lncZ reporter gene (line 1) or each of two multicopy 1ncZ reporters

Galactose 30°C

Glucose 30°C

Glucose 30°C

Figure 4. XTCl copurifies with the pol II holoenzyrne and is required for normal ce11

growth. a, Immonublot using anti-XTC1 antibodies of extracts (100 pg) from isogenic

wild type (lane 1) and xtc ld (lane 2) yeast cells and a portion (200 pg) of TFDS affinity

purified yeast pol II holoenzyrne (lane 3).b, Growth of isogenic wild type and xtcld yeast

strains on synthetic medium containing glucose at either 30°C or 37'C.

c, Impaired growth of xtcld cells on galactose and restoration of growth by ectopic

expression of XTC1. XTCl deficient cells were transformed with a vector expressing

XTCl from the ADHl promoter (pADH1-XTCI) or a control vector ( p A D H 1 ) . d , Growth

of isogenic wild type and x t c ld strains on glucose following overexpression of GAL4-

E2F-1 or the DNA-binding domain of GAL4 (GAL4DBD).

bearing either a strongly (line 2) or more weakly (line 3) GAL4 responsive promoter.

Surprisingly, GAL4 activated transcription from each of these reporter genes more

effectively in an xtcl A strain than it did in a parental strain. This increased activity of

GAL4 in w t c U cells was due to an enhanced activation potential of the C-terminal

activation domain of GAL4 since a LexA fusion bearing this domain was also more

active in wtc lA cells (line 6). As with deletions of genes encoding other components of

the pol II holoenzyme, namely SRBI, SRBZO, and SRB7 1 (ref. 17), deletion of the XTCl

gene synergized with a disruption of the MlGl gene to relieve glucose repression of

transactivation by GAL4 (line 5) compared with negligible relief of repression in

wildtype andxtcl A single mutant strains (not shown). Finally, transcriptional

activation by LexA-E2F-1 and, in particular, the mutant derivative of LexA-E2F-1

which bound poorly to XTCl was also markedly enhanced in xic2.A cells (lines 7

and 8).

Taken together, my results suggest that XTCl is a physioiogically relevant target of

activators which functions as a negative regulator of transcription. By binding to the

activation domain of activators XTCl may modulate the interaction of activators with

other components of the pol II transcriptional machinery. As overexpression of strong

activators inhibits pol II dependent transcription and impairs ceIl growthL582'8n, a

phenornenon termed " ~ ~ u e l c h i n ~ " ~ ~ (chapter 1), the growth defects ariçing frorn a

deletion of the XTCl gene may be due to the hyperactivity of one or more cellular

activators in the absence of XTCI. Consistent with this notion, I found that growth of

x k l A cells was dramatically impaired relative to wild type cells when a strong chirneric

activator, GAL4-E2F-1, was overexpressed (Fig. 4d) . Alternatively, like a number of other

components of the pol II transcriptional rna~h.i.ne$~-~~, XTCl rnay act as global

repressor of pol II transcription whose effects are partially relieved by direct contact with

activators. The lack of constitutive expression of a reporter gene in xtc ld cells in the

[ I I - 16

Activator Reporter p gai Units

Table 1. Hyperactivation of transcription in X ï C l defitient yeast.

B-galactosidase activities in permeabilized yeast cells transformed with the following lncZ reporter genes: lines 1, 4 and 5, the single copy integrated plasmid RY171 (ref. 22)

containing the GAL1 -IO UASg GALCbinding sites upstream of the GALl promoter;

line 2, a 2 pm derivative of RY171 (ref. 23); Iine 3, the 2 pm plasmid pJKïOl (ref 23), a

derivative of RY171 in which UASg has been placed a further 100 bp distal to the G A L l

promoter; lines 6-8, the 2 Pm plasmid pl840 (ref. 24) which has a single LexA operator

upstream of the GALl promoter. LexA-E2F-1 was expressed from the ADHI promoter in

a 2 pm vector. The expression vector for LexA-GAL4 (pSH17-4) has been d e ~ c r i b e d ~ ~ .

The strains used in lines 4 and 5 bore deletions of the GAL4 andMlGZ genes respectively. Cells were grown in 2% (w/v) galactose and either 2% (w/v) sucrose (lines 1-4 and 6-8)

or 2% (w/v) glucose (line 5), and were harvested at mid log phase. Activities are

expressed in standard unitsp; standard deviations were less than 20%.

I I I - 17

absence of an activator (Table 1, line 4) suggests, however, that the ability of activators

to interact with positive-acting components of the pol II transcriptional machinery,

such as TFIID, TFIIB, and TFIIH (refs. 3-5), may also be essential for activator function.

I expect that as protein microsequencing techniques become more sensitive, it will be

possible to identify additional protein targets of activators using the crosslinking

technique described here. Elucidation of the range of protein interactions mediated by

activators ris well as the mechaniçrn by which XTCl represses transcription should

lead to a more complete understanding of the regulation of transcription by pol II.

ACKNOWLEDGEMENTS

1 thank R. Kobayashi for performing the protein microsequencing, R. Brent, H. Ronne,

J. Archambault, and A. Pearson for generously providing plasmids, D. Jansma for help

in tetrad analvsis, and B. Andrews, J. Archambault, and J. Greenblatt for helphl

discussions.

I I I - 19

REFERENCES

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

Struhl, K. Annu. Rex Genet. 29, 651-674 (1995).

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

Tjian, R., & Maniatis, T. Ce11 77, 5-8 (1994).

Struhl, K. Ce11 84, 179-182 (1996).

Emili, A., & Ingles, C. J. \. Biol. Clzern. 270, 13674-13680 (1995).

Flemington, E. K., Speck, S. H., & Kack , W.C. P r x . IW!. Arc!! Sci. U.S.A. 90, 6914- 6918 (1993).

Dorman, G., & Prestwich, G. D. Biochemistry 33, 5661-5673 (1994).

Emery, H. S., Schild, D., Kellogg, D. E., & Motimer, R. K. Gene 104, 103-106 (1991).

Gorbalenya, A. E., Koonin, E. V., Donchenko, A.P. & Blinov, V. M. Niicl. Acids Res. 17, 4713-4730 (1989).

Ma, J., & Ptashne, M. Ce11 48, 847-853 (1987).

Sadowski, I., Ma, J., Triezenberg, S., & Ptashne, M. Nnture 335, 563-564 (1988).

Cress, W. D., & Triezenberg, S. J. Science 251, 87-90 (1991)

Koleske, A. J., Buratowski, S., Nonet, M., & Young, R. A. Cc11 69, 583-894 (1992).

Candau, R., & Berger, S. L. \. Biol. Chem. 271, 5237-5245 (1996).

Neigebom, L., & Carlson, M. Genetics 108, 845-858 (1984).

Balciunas, D., & Rome, R. Niicl. Acids Res. 23, 4421-4425 (1995).

Yocum, R. R., Hanley, S., West, R., Ir., & Ptashne, M. Mol. Cell. Biol. 4, 1985-1998 (1984).

Brent, R., & Ptashne, M. Ce21 43, 729-736 (1985).

Allison, L. A., & ingles, C. J. Proc. Natl. Acnd. Sci. U.S.A. 86, 2794-2798 (1989).

Gill, G., & Ptashne, M. Nature 334,721-724 (1988).

Berger, S. L., Pina, B, Silverman, N., Marcus, G. A., Agapite, J., Regier, J. L., Triezenberg, S. J., & Guarente, L. Ce21 70,251-265 (1992).

Yeung, K. C., Lnostroza, J. A., Mermelstein, F. H-, Kannabiran, C., & Reinberg, D. Genes Devi. 8, 2097-2109 (1994).

Merino, A., Madden, K. R., Lane, W. S., Champoux, J. J., & Reinberg, D. Nntrlre 365,227-232 (1993).

Auble, D. T., Hansen, K. E., Mueller, C. G. F., Lane, W. S., Thorner, J., & Hahn, S. Gerzes Devl. 8, 1920-1934 (1994).

He, Z., Brinton, B. T., Greenblatt, J., Hassell, J. A., & ingles, C. J. C d 73, 1223-1232 (1993

Poon, D-, Campbell, A. M., Bai, Y., & Weil, P. A. \. Biol. Chem. 269, 23135-23140 (1 994).

Kim, Y.+, Bjorklund, S., Li, Y., Sayre, M. H., & Komberg, R. D. Cell 77, 599-608 (1994).

Wang, R., Kobayashi, R., & Bishop, J. M. Proc. Nntl. Acnd. Sci. Lf.5.A. 93, 8425-8430 (1996).

30. Gietz, D., St. Jean, A., Woods, R. A., & Schiestl, R. H. Nzicl. Acids Res. 20, 6 (1992).

CHAPTER IV

Interaction of the C-terminal Domain of the Largest Subunit of RNA Polyrnerase II

with the Essential Splicing Factor PSF and the Putative Splicing Factor p54nrb

This chapter represents preliminary work still in progress.

(Except for the protein microsequencing, 1 did al1 of the experiments in this chapter tvith sorne technical assistance from M. Shales)

I V - 1

SUMMARY

1 have used affinity chromatography to characterize human proteins

which interact with the unique and essential C-terminal domain ( C m ) of the

largest subunit of RNA polymerase II (Pol II). Using this approach, 1 found that

the CTD interacts in a highly selective manner with several cellular proteins.

Two of these proteins, namely PSF and p54"rb, have been implicated

previously in the splicing of messenger RNA. 1 suggest that the interaction of

the CTD with these and perhaps other components of the splicing machinery

may be critical for coupling RNA processing to RNA synthesis by Pol II.

INTRODUCTION

The remarkable evolutionary conservation of the CTD of Pol II suggests

that it might serve as a docking site for cellular factors which function in key

aspects of Pol II-mediated transcription. In principle, these factors may be

involved in modulating the activity of Pol II at several different stages in the

transcription cycle. For example, one set of interactions may be cmcial for

transactivator-mediated recruitment of the Pol II transcriptional machinery to

a promoter. Consistent with this notion, the CTD has been shown to interact

in vitro with several different components of the Pol II transcriptional

machinery, such as the general transcription factors TFIID (Usheva et al. 1992)

and TFTIE (Maxon et al. 1994; Kang et al. 1995), as well as with a complex,

termed the 'mediator', of transcriptional cofactors (Thornpson et al. 1993; Kim

et al. 1994). A second set of CTD interactions need not, however, be directly

linked to the regulation of the initiation of transcription. For example, one

attractive possibility is that the CTD interacts with cellular factors which are

involved in the processing of nascent RNA transcripts.

In order to further characterize interactions rnediated by the CTD, I used

affinity chromatography to purify human proteins which interact specifically

with the CTD in vitro. In this manner, I have identified the essential splicing

factor PSF and the putative splicing factor p54nrb as CTD-interacting proteu-is.

This observation suggests that the CTD may be directly involved in coupling

messenger RNA processing to ongoing transcription though interactions with

the splicing machinery.

Expression and purification of recombinant proteins

A n E. coli expression vector encoding the complete CTD sequence of the

largest subunit of mouse pol II (a near perfect homolog of the human CTD)

fused to an N-terminal poly(l0)histidine tag was constructed in severai steps.

First, a Bgl II restriction site was introduced at the EcoRI site flanking the C-

terminal end of the CTD reading frame in the vector pGCTD (a kind gift of W.

Dynan). The 1.1 kb BamHl to BglII fragment was then subcloned into a

modified version of the expression vector pET19b (Novagen) in which the

BamHl reading frarne had been shifted first by blunting and religating of an

adjacent XhoI restriction site. The E. coli expression vector for N-terminal

poly(l0)histidine-tagged PSF was a generous gift of J. G. Patton (Vanderbilt

University). Complementary DNA encoding the complete open reading frame

of p54nrb (a kind gift of A. Krainer; CSHL) was subcloned for expression into

the Ndel and BamHl sites of pET19b. The proteins were expressed in the

E. coli strain BL21(DE3) and were purified by nickel chelate chromatography as

previouslv described (Emili and Ingles 1995). To synthesize [35S]methionine-

labelled protein derivatives, plasmids encoding the full-length cDNAs for

human PSF and p54*rb were transcribed in vitro and translated by a coupled

transcription/ translation system (Promega). The CTD was detected on a

Western blot using the monoclonal antibody JEL252 (Moyie et al. 1989) and

enhanced cherniluminescence (Amersham).

Affinity chromatography

HeLa ce11 extracts were prepared according to published methods

(Dignam and Roeder 1985; Shapiro et al. 1986) and had a final protein

concentration of -10 mg/ ml. Detailed procedures for affinity chroma tography

have been described (Emili et al. 1994). The purified CTD was coupled to

AffiGel-10 column matrix (BioRad) at the indicated concentration. For micro-

affinity chromatography, 300 pl of HeLa whole ce11 extract was

chromatographed through 20 pl affinity colurnns containing various

concentrations of the CID. The columns were washed with either 200 ul or

400 ul of affinity column buffer (ACB; 20 rnM Hepes-NaOH, pH 7.9,20 %

glycerol, 0.1 mM EDTA, 1 mM DTT) containing 0.1 M NaCl and eluted with

70 pl of ACB containing 1.0 M NaCl. For affinity chromatography with 3%-

labelled PSF and p54*rb, a 10 pl volume of each of the in vitro synthesized

radiolabelled proteins was mixed with 20 pl of ACB containing 0.1 M NaCl

and was loaded onto a microcolumn containing 20 pl of either matrix

containing 1 rng/ml immobilized CTD or matrix alone. The columns were

washed with 200 pl of ACB containing 0.1 M NaCl and were eluted with 70 pl

of ACB containing 1.0 M NaCl. One half of the volumes of the input and

eluate fractions were resolved by SDS-PAGE and detected by fluorography.

Purification and amino acid sequence determination

A 40 ml aliquot of HeLa whole ce11 extract was pre-passaged through a 2

ml affinity column containing ligand-free AffiGel 10 matrix alone. The flow-

through fraction was then applied to a 1 ml affinity colurnn containing 2

mg/ml CTD ligand. After washing with 10 column volumes of loading buffer,

the column was eluted with ACB containing 1M NaCl. The eluate was then

dialyzed extensively against ACB containing 0.1 M NaCl and

rechromatographed on a fresh 1 ml affinity column containing 2 mg/ml

immobilized CTD. This second column was washed with 10 colurnn volumes

of ACB containing 0.1 M NaCl and eluted with 4 ml of ACB containing 1M

NaCl. The peak protein-containing fractions (2ml) were pooled and

precipitated by the addition of one fifth volume each of 0.15% sodium

deoxycholate and 70% TCA. The proteins were resolved on a 10°h-SDS-

polyacrylamide gel and were visualized by staining with 0.05% ultra pure

Coomassie Brilliant Blue G-250 (Sigma) in 10% (v/v) Acetic acid and 25%

(v/v) methanol. The gel fragments corresponding to the stained bands were

excised and treated iit situ with Lysil-endoproteinase K (R. Kobayashi, Cold

Spring Harbor Labora tories, Microsequencing Facili ties). Peptides were

resolved by reverse phase HPLC and N-terminal amino acid sequence

determination was performed using an Applied Biosystem 475A protein

sequencing system (Wang et al. 1996). PSF and p54*rb were detected on

Western blots using immune serum generously provided by J. G. Patton

(Vanderbilt) and A. Krainer (CSHL) and enhanced cherniluminesence

(Amersham).

In Vitro kinase assay

The reactions were performed in 20 pl of kinase buffer (5 mM HEPES-

NaOH, 20 mM Tris-HCL, 7 mM MgC12,50 mM KC1,12% glycerol, 2% (W/V)

PEG 4000,0.5 mM DTT, 0.1 mM EDTA, 10 pM ATP, 6 pg of GST carrier protein,

and 2.5 pCi of [ y 3 2 ~ ] ~ T P (6000 Ci/ mmol; N'EN) for 1 hr at 25°C. The proteins

were precipitated with 1/10 volume each of 0.15% sodium deoxycholate and

100% trichloroacetic acid and were fractionated on a 12.5°/~-polyacrylamide gel

containing SDS. The gel was dried and exposed to film with a single

intensifying screen for 10 hrs at -70°C.

RESULTS

Preparation of a CTD Affinity Column

In order to identify human proteins that interact with the C-terminal

domain of Pol II, 1 expressed and purified a recombinant form of the mouse

CTD (a near perfect homolog of human CTD) in sufficient quantity and purity

for use a s an affinity ligand in a series of in vitro binding experiments. Since

the heptapeptide repeat sequence of the mouse CTD (Fig. 1A) was known to be

difficult to generate in sufficent quantities for use as an affinity ligand when

expressed as a GST fusion (W. Dynan, Univerity of Colorado; persona1

communication), I attempted to overexpress this domain in bacteria as a novel

fusion containing an N-terminal poly(l0)histidine sequence tag (Fig. 1B). This

form of the CTD was abundantly expressed in soluble form in E. coii cells and,

furthermore, could be purified to near homogeneity by single step nickel-

chelate affinity chromatography (Fig. 1C). Micro-affinity columns were then

prepared b y coupling the purified CTD covalently to an affinity column

matrix.

Affinity Chromatography

Soluble whole ce11 extracts were prepared from a human HeLa ce11 line

and applied to a series of affinity columns containing an increasing

concentration of immobilized CTD ligand. After loading the columns wiih

extract and washing extensively with buffer containing 0.1 M NaCl, the affinity

columns were step eluted with a high salt (1.0 M NaCl) buffer. The resulting

eluates were analyzed by SDSPAGE followed by silver staining of the gel. As

seen in Figure 2A, several proteins with apparent molecular masses of 180, 97,

KOrm a y tly N a ht kr P r o 3.r 1 T y r k r P r o T h r t r r P r o * L . 2 Tyr Glu P m lup kt P r o Cly G f y 3 ~ y r nar P r o bta Sor P r o kr 4 ~ y r k r P r o ~ $ u ? m â u S Tyr S a r P m Thr kr P m S a r 6 Tyr k r P r o Th k r P m A 8 a 7 Tyr wr P r o Zhr kr P m 3.r 6 Tyr k r 9 m Th kt P r o 3.r 9 Tyr k r P m T h r k r P r o k r 10 +yr *r P r o Zhr 3.r P m âu Il Tyr k r P m Zhr k r P r o Ilu 12 T y r s u P r o T h r S u P m h r 13 fyt 5.r P m Thr k r Oro k r 14 Tyr k r P r o ftu kr P m k r 5 tyt wr 9 m Zhr Su P m 3.r t8 ~ y r S a r Oro Zhr k r P m Su 17 tyr r r p r o ~ h r k r ? m âu 18 Tyr Smr P m Zhr k r P m 5.r 19 Tyr k r P m Zhr k r Pro &r aI Tyr 5.r P r o Zhr trr Pro S o r n lyr SU s m ~ h r 5.r P m k r P ryr k r P r o Thr k r P m iua zl r y r s r r P r o i b r k r P r o A 8 a # syr tb.r p r o rb r s r P m %r a ryr sar P m Zhr Smr P m 3.r

ryr a r P m T h r k r P m A m 27 T y r I h r P r o ? h z S m r P m A 8 ~

fyr 5.r P m Zhr kr P m 3.r 29 Tyr M r P m Thr k r P r o Sa= JO Tyr Slr Pro Thr k r P r o Sor 3I Tyr k r Pro kr S.r: P m Irg a r y r Pro GLO k r P m % p Tyr Ihr Pro kr k r P r o Su 3 fyt Sor Pro kr k r P m k r 35 Tyr ?L.r Pro k r P m Ly8 S Tyr Tür P m Zhr k r P r o 3.r 37 Zyr Slr Pro kr k r P r o Glu 38 Tyr Tür Pro Zhr 3.r P m Ly8 1D Tyr 9ir P m Thr k r P r o Lya Q Tyr k r Pro Thr k r P m Ly8 41 Tyr 5.r P m Zhr kr P r o Thr 4 Tyr Sor P m nu P r o LYS 4 Tyr k r Pro n%r kr P m Thr U Tyr S a r P m Zhr 3U P r o V a i lb Tyr Thr Pro Thr kr P m Lys

Tyr wr P m Zhr Smr P m Thr 47 Tyr S a r P m Zhr k r P m Lyi 40 Tyr k r Pro Thr S u P m S o r Q Tyr a r P m Zhr 3.r P r o Ly8 Cly $or T k 9D Tyr k r P m Zhr kr P m W y 5ï Tyr k r Pro n%r kr P r o Thr Q Tyr S a r L m Thr kr P r o N a

r t . r a R o 4 y p ~ 4 Q n C L i ~ C ( r r m

66 - CTD -,

Coomassie stain

Western blot

Fig. 1. Expression of the CTD of mouse in recombinant form. A, amino acid

sequence of the CTD of the largest subunit of RNA polymerase II from mouse.

B, Schematic of the CTD bacterial expression construct showing the N-

terminal poly(l0)histidine tag and the T7 RNA polymerase promoter. Cr (Left

panel) Coommasie blue stained gel showing purified recombinant mouse CTD

(1 pg); (Right panel) Western blot of purified recombinant CTD (1 pg) using

the anti-CTD monoclonal antibody JEU52 (Moyle et al. 1989). M, of protein

markers are given at the left in kDa. The CTD is highlighted by an arrow.

80,60,40,36,34, and 32 kDa were found to bind specifically to the CTD affinity

columns but not to the control matrix alone. The yield of these proteins

increased in direct proportion to the concentration of the CTD ligand on the

column (compare lanes 1 to 5), emphasizing the specificity of these

interactions. Subsequently, 1 found that if a more extensive washing procedure

was employed prior to the high salt elution step in order to eliminate

background binding, the polypeptides which bound specifically to the CTD

affinity resin could be more readily visualized (Fig. 28, compare lanes 1 and 2).

This discovery of human CTD-binding proteins was particularly

exciting since it had been shown by others that the CTD interacts, at least in

yeast, with a large protein complex, termed the mediator, which contains a

number of the accessory transcriptional cofactors which regulate the activity of

the Pol II transcriptional machinery in vivo (Chapter 1). As at least one

component of the mediator complex encodes a protein kinase which can

specifically phosphorylate the CTD in vitro (Liao et ai. 1995), I assessed

whether the eluates of the CTD column contained a kinase activity capable of

phophorylating recombinant CTD. As seen in Figure 3, the eluate from a CTD

affinity column contained a robust CTD-kinase activity. Furthermore, this

kinase activity appeared to be specific for the CTD since several other control

proteins tested were not detectably phosphorylated (data not shown). As no

CTD kinase activity was detected in the eluate from a control column nor with

the recombinant CTD preparation alone (data not shown), it appeared that the

CTD-kinase was retained through specific association with the CTD.

Interestingly, Western blot experiments indicated that the CTD column eluate

did not contain significant quantities of the human general transcription

factors TFIID and TFIIE (data not shown) although each of these factors had

been proposed

I V - 1 O

I I

m CTD - CTD

Fig. 2. Affinity purification of CTD-binding proteiw from a HeLa ce11 extract.

A, Silver stained SDS-gel showing the protein profile of the high salt eluates

from a series of affinity microcolumns loaded with a portion of HeLa whole

cell extract. The concentration of coupled recombinant CTD ligand is indicated

above each lane. B, SDS-PAGE analysis and siiver staining of high salt eluates

from CTD-affinity columns (lanes 2 & 4) or control (ie no ligand) columns

(lanes 1 & 3) loaded with HeLa whole ceil extract. For lanes 3 and 4, the extract

was first pretreated with 0.1 mg of RNase A for 30 min on ice before being

loaded on the columns.

I V - 1 1

CTD Eh (Ci0

Fig. 3. CTD kinase activity in the eluate from a CTD affinity column.

A, Autoradiogram of SDSPAGE fractionation of in vitro kinase reactions

containing the indicated volume of high salt eluate form a CTD affinity

column loaded with HeLa celI extract. Recombinant CTD (1 pg) was added to

the reactions shown in the two right-most lanes.

previously to interact with the CTD (Usheva et al. 1992; Maxon et al. 1994).

1 did find, however, that a recombinant form of the TATA-binding subunit

(TBP) of TFTID was capable of interacting directly with the CTD ligand (data not

shown).

Identification of two CTD-binding proteins

To identify some of the proteins present in HeLa ce11 extracts which

bound specifically to the CTD, I scaled up the purification procedure in order

to obtain a sufficient amount of protein for direct microsequencing. T'he input

HeLa ce11 extract was first pre-cleared by passage through a column prepared

with uncoupled a f h i t y matrix alone, and the flow-through fraction from this

column was then bound to and eluted from two sets of CTD affinitv columns

in succession. Two major polypeptides of apparent molecular masses of 60 and

97 kDa were found to be selectively purified to near homogeneity (Fig. 4, panel

A). After preparative scale SDS-PAGE (Fig. 4B), the bands corresponding to

these two polypeptides were subjected to proteolytic cleavage in sihr

(R. Kobavasi, Cold Spring Harbor Laboratories, Microsequencing Facility). The

resulting peptides were resolved by reverse phase HPLC and subjected to N-

terminal (Edman) amino acid sequence determination. Good sequence

infornation was obtained for multiple peptides derived from both the 60 and

97 kDa polypeptides. These sequences were used to search the GenBank and

EMBL protein sequence data banks and were found to match perfectly (Fig. 4C

and D) to sequences encoded by the human RNA-binding protein PSF

(polypyrimidine tract-binding protein-associated splicing factor; Patton et al.

1993) and the human RNA-binding protein ~54nrb (nuclear RNA-binding

protein p54, also known as nonA/BJ6; Dong et al. 1993; Yang et al. 1993), a

PSF

Fig. 4. Purification and identification of two CTD-interacting proteins.

A, Silver-stained SDS-gel showing a portion of the most highly purified CTD-

binding protein preparation. B, Coomassie blue stained gel showing the

preparation of CTD-bùiding proteins submitted for microsequence analysis.

The protein bands corresponding to PSF and ~ 5 P b are highlighted by arrows.

C and D, amino acid sequence of the hurnan PSF and p~4mb polypeptides.

The residues corresponding to peptide sequences obtained from the

microsequence analysis are underlined.

protein which shares considerable homology to PSF. PSF is an essential factor

required for splicing of messenger RNA (Patton et al. 1993; Gozani et al. 1994).

p54"'b has also been suggested to be a splicing factor (Dong et al. 1993; Hallier et

al. 1996).

To verify that both PSF and p54mb were indeed capable of interacting

specifically with the CTD, we performed Western blotting experirnents on the

salt-eluted fraction from the CTD affinity column using polyclonal antibodies

generated against either PSF (Fig. 5A) or p54nrb (data not shown). A single anti-

PSF immunoreactive band with the same mobility a s the native form of this

protein was present in the eluate from the CTD column (lane 3) but not that of

the control column (lane 2). The specificity of this antibody was confirmed

using a recombinant PSF produced in E. coli as a positive control (lane 5 ) . A

similar result was produced in Western blotting using antbp54nrb serum (data

not shown). Therefore, 1 conclude that both PSF and p54nrb interact specificaily

with the CTD.

PSF and p54nrb

The apparent 1:1 stoichiometry of the PSF and p54nrb polypeptides in the

CTD affinity column eluates suggested that they form a heterodimeric

complex. Indeed, PSF and p54nrb do interact in a 2-hybrid assay (P. Tucker,

Univ. of Texas, Austin; persona1 communication). To determine which of

these individual polypeptides might mediate the interaction with the CTD, we

generated [3sS]-methionine-labelled derivatives of both PSF and p54nrb in a

rabbit reticulocyte lysate through individual translation of full-length cDNA

clones encoding each protein. Radiolabelled PSF and p54*rb were evaluated for

their ability to interact with immobilized CTD ligand. The input samples and

the materials eluted from a CTD affinity column and a control column were

fractionated by SDS-PAGE and analyzed by autoradiography (Fig. 5 ) . Both PSF

and, to a lesser extent, p54nrb were found to be selectively retained on the CTD

affinity resin suggesting that each protein is capable of interacting with the

CTD. Nonetheless, it was apparent that the affinity of the radiolabelled PSF

and p5Wb for the CTD ligand was substantially lower than that exhibited by

the native f o m s of these proteins in HeLa extract. This discrepancy suggested

that the ability of these proteins to bind efficiently to the CTD might be

dependent on their prior assembly as a complex or their isolation from HeLa

cells. We have not been able, however, to demonstrate a direct interaction

between the CTD and purified recombinant f o m s of either PSF and p54*rb that

were CO-expressed in bacteria and CO-purified as a complex (R. Gupta, persona1

communication). Therefore, it is possible that the interaction of cellular forms

of PSF and p54nrb with the CTD is mediated through an other (intermediate)

factor(s) or requires some post-translational modification(s) in either protein.

The CTD has the ability to interact with nucleic acids, albeit weakly, in

vitro. Also, both PSF and p54nrb have concensus RNA-binding motifs and are

capable of interacting in an high affinity manner with RNA substrates in oifro

(Patton et al. 1993; Yang et al. 1993; Gozani et al. 1994; Hallier et al. 1996). As

such, the interaction of native PSF and p54nrb with the CTD may be mediated

through a nucleic acid intermediate present in the ce11 extract. However, this is

unlikely since eluates from the CTD affinity columns did not contain

signifiant amounts of nucleic acid as detected in vitro by direct labelling with

T4 polynucleotide kinase (data not shown). Furthermore, extensive pre-

treatment of the input HeLa extract with large quantities of either RNase A

(Fig. 2B, lanes 3 and 4) or microccocal nuclease (data not shown) failed to

I V - 16

El coli Exttact

L A 1 cn - %

PSF

Fig. 5. Binding of PSF and p54mb to a CTD affinity column. A, Western blot of

an SDS-gel probed with anti-PSF serum. Lane 1, a portion of HeLa ce11 extract

loaded on the affinity columns; lane 2, high salt eluate from a control column;

lane 3, high salt eluate from a CTD affinity column; lane 4, extract prepared

from uninduced E. coli cells; lane 5, extract hom E. coli cells induced to express

recombinant full length PSF. B and C, Fluorograms of SDEPAGE fractionated

input protein (lane 1) and high salt eluates from a set of CTD affinity columns

(lane 2) and control columns (lane 3) loaded with either [35S]methionine-

labelled PSF (panel B) or p 5 P b (panel C).

IV- 17

impair the binding of native PSF or p54nrb to a CTD column. Furthermore, the

addition of exogenous poly-UMP resulted in the elution of both PSF and

p54nrb from a CTD column without the requirement for increased ionic

strength (data not shown), suggesting that the binding of PSF and p54nrb to the

CTD and RNA might be mutually exclusive.

I V - 18

DISCUSSION

Using an affinity chromatography assay, 1 have found that the splicing

factor PSF and the putative splicing factor p54nrb interact with the CTD. This

observation suggests the exciting possibiIity that the CTD may play a role in

coupling the process of RNA splicing to nascent production of mRNA. A

number of observations are consistent with this notion. First, structural and

biochemical studies have firmly established that the process of splicing of pre-

mRNA transcripts is temporally and spatially linked to Pol II transcription in

the ce11 nucleus (Jimenez-Garcia and Spector 1993; Matunis et al. 1993; Weeks

et al. 1993; Bauren and Wieslander 1994; Richler et al. 1994; Zhang et al. 1994;

Bregman et al. 1995; Mortillaro et al. 1996). For example, immuno-

histochemical staining of hurnan nuclei indicates that splicing occurs

exclusivelv at sites of active transcription (Jimenez-Garcia and Spector 1993;

Zhang et al. 1994). Second, it has become apparent that assembly of an active

spliceosome complex occurs through the sequential association of components

of the splicing machinery with the nascent pre-mRNA transcript (Beyer and

Osheim 1988; Amero et al. 1992; Matunis et al. 1993; O'Keefe et al. 1994;

Wuarin and Schibler 1994; Huang and Spector 1996). Third, it was found that

mRNA transcripts are not spliced if transcribed by RNA polymerase i or III

(Sisodia et al. 1987; White and Kunkel 1993). Fourth, it was found that the

addition of a CTD-like repeat oligopeptide to in vitro splicing reactions

specifically inhibits splicing of pre-mRNA substrates (Yuryev et al. 1995).

Finally, i t was shown recently that truncation of the CTD greatly impairs pre-

mRNA processing in i?iz70 (McCracken et al. 1996, submitted) and that the

CTD interacts with a number of other pre-mRNA processing factors, in

particulaï certain novel members of the SR family of splicing cofactors, in

addition to PSF and p54nrb (Yuryev et al. 1995; McCracken et al. 1996,

submitted).

How might the interaction of the CTD with PSF and p54nrb regulate

splicing? Like transcription, pre-mRNA splicing is a highly regulated process

which involves the coordinated activity of a large number of proteins

(reviewed by Green 1991; Lamm and Lamond 1993; Rio 1993; and Newman

1994). Antibody inhibition, immunodepletion, and biochemical reconstitution

studies have indicated that PSF functions at several stages during the

formation of an active splicesome complex which, in tum, mediates the

processing of an RNA transcript (Patton et al. 1993; Gozani et al. 1994). PSF

interacts with the polypyrimidine tract of mammalian introns (Patton et al.

1993; Gozani et al. 1994), an element located adjacent to the branchpoint and 3'

splice acceptor sequences which is known to modulate the efficiency of splice

site usage (Green 1991; Mullen et al. 1991; Patterson and Guthrie 1991; Lamm

and Lamond 1993; Rio 1993; Roscigno et al. 1993; Newman 1994). Therefore,

PSF may be involved in mediating recognition of the 3' intron boundary.

Through its interaction with PSF, the CTD may also influence splice site

selection by targetting both this and other components of the splicing

machinery to specific intron-exon sequences. Iriterestingly, p54nrb has also been

found to interact directly with a number of sequence-specific DNA-binding

proteins (Hallier et al. 1996; J. Hassel, McMaster University, personal

communication) in addition to interacting with the CTD. This suggests that

p 5 4 ~ b could serve to integrate signal transduction pathways with the CTD and

the splicing machinery. Conversely, as the disruption of splicing leads to a

generalized impairment of transcription by Pol II iri vivo (OIKeefe et al. 1994),

the link between splicing factors and the CTD may also play some role in

regulating transcription by Pol II.

In order to evaluate the role of the CTD in the processing of pre-mRNA

transcripts, 1 have been atternpting to develop a transcription-dependent, or

coupled, i r l vitro splicing system. This biochemical approach to the study of

CTD function in mRNA processing has certain advantages over the studies on

CTD function performed in vivo since it should be possible to establish

conditions which bypass the requirement for the CTD in the initiation of

transcription. A combination of this and other biochemical and genetic

approaches will be necessary to elucidate the physiological function of the CTD

in RNA processing.

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93:8425-8430.

Weeks, J. R., Hardin, S. E., Shen, J., Lee, J. M., and Greenleaf, A. L. 1993. Locus-

specific variation in phosphorylation state of RNA polymerase II in vivo:

correlations with gene activity and transcnpt processing. Genes D e n 7:2329-

2344.

White, R. A., and Kunkel, G. R. 1993. Pre-messenger RNA splicing of

transcripts synthesized from human small nuclear RNA gene promoters.

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transcribed by RNA polymerase II: evidence for cotranscriptional splicing.

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2 3:5593-5603.

Yuryev, A., Patturajan, M., Ltingtun, Y., Joshi, R. V., Gentile, C., Gebara, M.,

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Zhang, G., Taneja, K. L., Singer, R. H., and Green, M. R. 1994. Localization of

pre-mRNA splicing in mammalian nuclei. Nntzire 3739309-812.

APPENDIX

The RNA Polymerase II C-Terminal Domain:

Links to a Bigger and Better 'Holoenzyme'?

Andrew Emili and C. James Ingles

ïhis review was published in Current Opinion in Genetics and Development

Vol 5, April 1995

SUMMARY

The largest subunit of eukaryotic RNA polymerase Ii has an

unusual tandemly repeated heptapeptide sequence at its carboxyl-terminus.

The function of this evolutionarily conserved C-terminal domain is not

known. New evidence, however, links it to the formation of a large multi-

component RNA polymerase II complex possessing enhanced transcriptional

initiation properties. The existance of a preassembled RNA polymerase II

'holoenzyme' in the ce11 calls into question the long held view of

transcription initiation as an ordered promoter-dependent process.

INTRODUCTION

A major new character, the 'holoenzyme', has emerged in recent

studies of RNA polymerase II (Pol II), the enzyme responsible for the synthesis

of messenger RNA in eukaryotes. Holoenzyme is the name given to two

related forms of Pol II in which the 12 subunit core enzyme is found associated

with a number of accessory transcription factors [1@.,2a .]. Unlike the minimal

core enzyme, which permits only a low basal level of transcription i ~ c z~itro,

these high molecular weight Pol II complexes can mediate a response to

transcriptional activators in vitro and therefore may represent a form of Pol II

more like that which functions in the cell. Several genetic and biochemical

criteria tentatively suggest a role for the unique carboxyl-terminal domain, or

CTD, of the largest subunit of Pol II in both the assembly and the activity of the a

holoenzyme. The existence of a preassembled cellular pol II transcription

complex challenges previous concepts of transcription initiation as an ordered

multi-step assembly process and may provide new clues as to how the activity

of Pol II is regulated iu vivo. This review will focus on the biochemical

properties of two forms of pol II holoenzyme characterized in the past year and

the implications of their discovery to Our understanding of Pol II mediated

transcription.

The basic transcriptional machinery

There has been significant progress in recent years in elucidating the

fundamental mechanisms that govern transcriptional initiation by pol II. Al1

of the protein factors required for basal, activator-independent initiation of

transcription in in vitro systems have now been purified and, in most cases,

their corresponding genes have been cloned. Pol II can initiate transcription

from a promoter in vitro in the presence of five factors, namely the TATA-

binding protein TBP, TFIIB, TFTIE, TFIIF, and TFIIH [3,1]. This ability to

reconstitute accurate initiation in vitro using a fairly well defined set of

proteins has in turn led to models detailing how sequence-specific DNA-

binding transactivators might determine Pol II initiation rates in i?ii?o. Since

the assembly of a preinitiation complex at a promoter has often been viewed

as a sequential process [3,4], transcriptional activators are thought to stimulate

the rate-limiting steps in this overall pathway [5,6]. in in vitro assays

containing TBP and the other general initiation factors, however,

transcription by Pol II is not intrinsically responsive to transactivators [2. *,7].

Several candidate accessory proteins, or coactivators, likely to be required for

activator-mediated stimulation of transcription have been identified by a

combination of biochemical and genetic approaches. One set is the TBP

associated factors or TAFs, which, together with TBP, make up one important

target of transactivators called TFIID. Another source of coactivator function

now appears to reside in components of the Pol II holoenzyme.

Suppressors of CTD mutations

The evolutionarily conserved carboxy-terminal domain (CTD) of the

largest subunit of pol II consists of an array of near-perfect repeats of the

sequence YSPTSPS reiterated 26 times in yeast [SI and 52 times in mammals [ 9 ] .

The CTD is not found in the otherwise quite similar largest subunits of RNA

polymerase 1 and III or in their homolog P' in E. coli RNA polymerase [IO].

Although essential for ce11 viability [ll-141, the CTD is not required for

initiation by Pol II in a simplified in vitro transcription system reconstituted

with purified general initiation factors [14-161. The CTD may be linked in some

way, however, to the regdatory mechanisms that control initiation by pol LI

since it can influence the response of the enzyme to transcriptional activators

both in vivo [17,18] and in vitro [19]. Partial truncation of the CTD can lead to a

range of conditional growth phenotypes in yeast including temperature

sensitivity and nutrient auxotrophy [20-22.1, phenotypes probably due to

defects in the transcription of certain essential genes. This property was

exploited in the R.A. Young laboratory where four different dominant

extragenic suppressors (SRBZ, SRB4, SRB5, and SRB6) of yeast strains bearing a

partially deleted CTD were isolated [20-22.1. The proteins encoded by these

SRB genes may be candidates for a new class of transcription factor since

deletion oi either SR82 or SRB5 displayed a growth deficiency similar to that

exhibited by the CTD truncation [20-22.1. Although the predicted amino acid

sequence encoded by the four dominant-acting SRB alleles cloned to date has

not revealed any dues as to their function, an analysis of in vitro transcription

using yeast ce11 extracts suggested that at least several of the SRB proteins

contribute to the formation of a pol II preinitiation complex [21,22*].This effect

might be mediated through direct or indirect interaction of one or more of the

SRB proteins with the CTD since it was found that they could be selectively

retained on a CTD affinity column [22@].

Enter the holoenzyrne

In an attempt to further characterize the role of the SRB proteins in

transcription, the Young group choose to purify these factors from a yeast ce11

extract using conventional chromatography. Intriguingly, the four SRB

polypeptides were found to copurify within a large complex that included the

12 core subunits of Pol II [22.] and several additional polypeptides. Initially,

only 2% of the cellular pol II was estimated to be in this complex, however,

suggesting it was a minor form of Pol 11 in the cell. TBP was also initially

reported to copurify with these SRB proteins [22.] , an observation that

appeared to tie in nicely with previous dernonstrations that TBP could bind

directly to both the CTD [23] and SRB2 and SRB5 [21,22.]. It now seerns,

however, that TBP, and the link it might provide between the CTD and the

SRB proteins, is progressively lost from the complex during purification [l*.].

Nonetheless, the highly purified pol II-SRB complex required only TBP and

TFIIE to initiate transcription from a promoter in z~itro and, as expected

therefore, the complex was shown to contain the three other essential

initiation factors, TFIIB, TFIIF, and TFIM. Although combinations of these

factors and Pol II had been shown previously to bind one another iii idro [3,4],

the existance of a preassembled Pol II cornplex, or holoenzyme, came as a

surprise since much work had contributed to the idea that these factors

associate only at a promoter in a series of steps coordinated by transcriptional

activators (see Fig-IA). This raises a concem that the holoenzyme might

represent Pol II released from initiation complexes present at promoters

during ce11 breakage or, altematively, a form of Pol II engaged in elongation.

The latter possibility appears unlikely, however, since neither TFIIB or TFIIH

are thought to associate with the polymerase during elongation [4,24]. A

signifiant feahire of the holoenzyme was its ability to support activator-

dependent transcription iiz vitro suggesting that several of its associated

factors, including the SRBs, might be important for the activation process in

vivo.

A 'mediator' of transactivation

A somewhat different and much more abundant form of Pol II

holoenzyme was discovered in the R. D. Komberg laboratory [Ze.]. In the

course of purifying the yeast general initiation factors, this group came upon a

loosely defined nuclear fraction, termed the 'mediator', that could relieve the

transcriptional inhibition or 'squelching' observed when large amounts of a

strong transactivator are added to in oitro transcription reactions [El. This

same mediator also permitted a response by Pol II to activators such as GAL4-

VP16 and GCN4 in a partially purified in vitro system [26]. Although these

preliminary experiments suggested a coactivator role for the mediator in

transactivation, it was not clear if it exerted its effects in an indirect marner

such as by countering the effects of non-specific inhibitors of transcription. A

major advance, therefore, was the purification of a single complex of about 20

polypeptides that permitted a response to GAL4-VP16 and GCN4 in an in vitro

transcription system reconstituted with essentially pure general initiation

factors and Pol II [2. m l . This new mediator differed Çrom the first reported by

this labora tory in that i t also strongly stimulated activa tor-independent

transcription. Consistent with this stimulatory activity of basal transcription,

the new mediator was found to contain the four SRB proteins characterized by

the Young group and found in their holoenzyme [ la.] . It also contained

GALl1, a protein involved in both basal transcription [27] and in the

activation of transcription by GAL4 and other activators [28], and SUGl,

another protein implicated in the response to GAL4 [29].

One important aspect of this new mediator was that it could also be

copurified in a complex with Pol II [2* . ] . This form of Pol II, reportedly

accounting for at least half of the cellular Pol 11, was also termed the

'holoenzyme' by the Kornberg group. The mediator could be resolved from

the core enzyme by immunoaffinity chromatography on an anti-Pol II CTD

antibody column [2@ a]. This suggests, albeit indirectly, that the CTD may

provide a physical link between the core Pol II enzyme and the mediator

complex. Importantly, a holoenzyme complex responsive to transcriptional

activators in vitro, could be reconstituted with core pol II and the purest

media tor prepara tion. One notable difference between this holoenzyme

complex and the one isolated in the Young laboratorv [l-] is that it contains

only one of the general initiation factors, TFIIF, and not TFW, TFIIH, nor any

TBP. This marked difference in composition may be a consequence of the

significantly different strategies used to purify the two complexes. If this is the

case, it rnight be reasonable to expect that even larger assemblies of Pol II

initiation and regdatory factors exist in the cell. Indeed, a h l ly preassembled

Pol II transcription complex rnight be the predominant form of Pol II that

responds to activators in vivo (Fig.lB). Nonetheless, the observation that a

holoenzyme form of Pol II can support activator-dependent transcription

using TBP differs from other studies in both yeast [30] and mammalian

systems [7] in that there does not seem to be any requirement for the TBP-

associated proteins or TAFs. Although SRB2 and SRB5 have been reported to

bind to TBP [21,22a], none of the SRBs appear to be the yeast homologs of the

mammalian TAFs [30]. The holoenzyme complexes also do not require TFIIA

for response to activators even though several recent studies have shown

TRIA to be essential for activator-dependent transcription [31,32]-

Transcriptional activation in vitro is notoriously condition dependent,

however, and it is likely that the activation observed with the holoenzyme i r l

vitro is only a partial response. On the other hand, truncation of the CTD

appears to be more deleterious for the transcription of some genes than others

[18], suggesting that certain activators might function in a mediator-

independent manner.

Loose connections

The evidence accumulated so Car suggests a role for the CTD in the

assembly and activity of the Pol II holoenzyme. Certain aspects of this

hypothesis can now be tested. For exarnple, one prediction is that a Pol II

enzyme lacking the CTD might not form a stable complex with the mediator

complex and should, therefore, be unresponsive to activators such as GAL4-

VP16 and GCN4 in oitro. Even then, a major issue to be resolved is how

components of the mediator facilitate the response of pol II to transactivators.

One possibility is that, in conjunction with TFIIE and TFIM [33,34], they aid in

the formation of an open preinitiation cornplex or in promoter clearance by

Pol II, two steps in the initiation of transcription that can be stimulated by

transcriptional activators [35,36]. SRB proteins with a dominant gain-of-

function suppressor mutation [20-22.1 rnay be sufficiently active in this process

such that they bypass the requirement for activators and/or a wild-type length

of CTD on Pol II at certain promoters. Alternatively, as the growth defect due

to a CTD truncation can also be relieved by a nul1 mutation in the S N 1 gene

[37], which encodes a putative component of chromatin, the mediator may

also be playing a role in the remodelling of nucleosome stucture around

transcribed genes. Neither of the two Pol II holoenzymes described to date,

however, appears to contain the SWI/SNF complex of regulatory proteins that

are thought to catalyze this process although one component of the SWI/SNF

complex may also be part of the holoenzyme [38]. Intriguingly, SUG1, a protein

associated with the mediator, was also found recently to be an intrinsic

component of the 26s proteasorne [39], the major cellular degradation

machinery for ubiquitin-tagged proteins. It is not clear if SUGl is serving in

two very different cellular processes or if it links these two processes in sorne

manner much like TFIIH, which functions in both transcriptional initiation

and nucleotide excision repair [24]. Although the identity of a nurnber of

additional polypeptides in the holoenzyme has not yet been reported, one can

anticipate that they too may be novel transcription regulatory factors.

Another issue not yet addressed in studies of the Pol 11 holoenzyme is

the role of phosphorylation of the CTD, itself a subject of several recent

reviews [24,40]. Phosphorylation of the CTD is linked to the transition from

the initiation to elongation phase of transcription [41-431 and may therefore

play a role in the disassembly of the holoenzyme. In this respect, it is

interesting to note that the mediator can stimulate the activity of a CTD kinase

associated with TFW [ 2 * * ] . As T F W is a target for direct binding by activators

[44] it is possible that activators might also influence phosphorylation of the

CTD. Although phosphorylation of the CTD is not essential for basal [45] or

activated [46] transcription in vitro, it is probably important in regulating some

aspect of the activity of Pol II in vivo.

The remarkable conservation of the CTD throughout evolution

deserves additional comment. Were an ancestral CTD required to make

contact with two or more factors, then retention of the YSPTSPS sequence

would be subject to unusually high selective pressure. The factors that interact

with the CTD may even be involved in different functions. One set of factors

might be components of the holoenzyme. A second set of CTD interactions

need not be linked to transcriptional initiation. One attractive idea is that the

CTD plays a role in splicing of nascent transcripts [47a], a process almost

exclusively associated with Pol II. Although the colocalization of splicing

components and Pol II with a hyperphosphorylated CTD has been reported

[43], additional genetic and biochemical experiments rnight provide more

definitive evidence for the involvement of the CTD in hnRNA processing or

other cellular activities.

Conclusions

Transactivators appe ar to stim ulate transcription b y interacting with

multiple cornponents of the Pol II transcription initiation machinery (see

review by Treizenberg in this issue]. Figure 1A (Chapter 1) shows a version of

the classic multi-step model for activation of Pol II mediated transcription. In

this model, a sequence-specific activator is thought to hasten the assembly of

preinitiation complex at a promoter by interacting with distinct Pol II

initiation factors at multiple discrete steps. One prediction from this model is

that each interaction mediated by the activator is likely to be crucial for the

productive assembly of the initation complex. A n alternate interpretation of

the many studies that have detailed this ordered recruitment of individual

factors to a promoter. however, is that it merely reflects the catalog of

individual protein-protein contacts that occur within a large holoenzyme

complex. The speculative model shown in Figure 18 (Chapter 1) suggests that

an activator recruites a preassembled holoenzyme complex in a single step.

This holoenzyme pathway to the initiation of transcription may allow some

redundancy in the number of interactions between activators and the

holoenzyme needed to influence initiation. Establishing if this more simple

holoenzyme pathway of transcription initiation iç the one that operates in

vivo will be a difficult but crucial task for the future.

ACKNOWLEDGEMENTS

We thank M Shales for the preparation of the figure. Research in the authors'

laboratory is supported by grants from the Medical Research Council of Canada

and the National Cancer hstitute of Canada.

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and linking this process to RNA synthesis.

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