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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
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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).
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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).
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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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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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processing thereby facilitating the splicing of nascent pol II RNA transcripts
and linking this process to RNA synthesis.
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