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 National Library Bibliothèque nationale I*B of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. me Wellington Ottawa ON KIA ON4 Otmm ON K1A ON4 Canada Canada Yovr Me Votre reiémœ
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The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. 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 additional 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. ..* Ill.
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, it 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.
Preface r. Promoters of Transcription 11. Sequence-Çpecific Activa tors of Transcription DI. The Pol II Transcription Cycle . IV. The CTD . v. 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. ME,TFIIF, and TFIIH ii. Transcriptional Cofactors . a.TheMediator . b. The SWI/SNF Complex c. Adaptors . d. TFIIA e. Elongation Factors VI. Models of Transcriptional Activation . VII. Closing Comments VIII. Thesis Rationale . IX. 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 . viii.
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 IV40 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
II- 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 iil 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 (zl kb) distal to a target promoter. However, in the majority of cases these regulatory sequences are usually found adjacent (~400bp) 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 as 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 Kd 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 replation 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 et 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 (eg.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 irl oitro into six sequential steps which are defined operationally as (1)promoter commitment, (2) open
complex formation, (3) initiation of transcription, (4) promoter clearance' (5) 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~~ZJO 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 NAchain 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 fom 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 Cmimpairs 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 on 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 irr 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 oih 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 et al. 1993; Purnell et al. 1994; Kauham 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 et 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 et 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 in 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 cm 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 cm result in activated levels of transcription in oioo in the absence of a bone fide activation domain (Chatterjee 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 1993; Auble et al. 1994), and HMGl (Ge and Roeder 1994), which appear to inhibit the formation of a preinitiation complex by interacting directly with TBP. 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 iri 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 irl 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 iri 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), it 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 iii 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 fom of the CTD (Maxon et al. 1994; Kang and Dahus 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). It 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 TFW 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, PIIE 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~itro(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 hction 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~zi7iij0 (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~ivitro 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 in i~iiio is impaired in strains bearing a deletion of SRBlO/lI 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 SRBS, 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 1-28
con tains at least six gene products 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 iil 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-promotercomplex can be a rate-limiting step in the initiation of transcription in ~itrowhich 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~itro dramatically reduce activation of transcription iii oitro and iii i~ii70 (Ozer et 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 ~itro,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 +n-lv; TATA 0 I TATA
TATA t
TATA 7 4 Stepwise Recruitrnent 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~im(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. REFERENCES
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Zhu, A., and Kuziora, M. A. 1996. Homeodomain interaction with the beta subunit of the general transcription factor TFIIE. 1. Biol. Clienz. 271:20993-20996. 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 fom a preinitiation complex (reviewed in Ref. 1; Chapter 1). Sequence-specific 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~itro.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
PIID (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 the 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 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 chah 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 TR 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~toethanoland 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 at 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 [y32~]~~~ (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~-labeledTEP 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 KI,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 ATand UTP, 20 pM CTP, 20 units RNase Block 1 (Stratagene), 10 units RNaseTl (Boehringer), 2.5 pCi [~~~P]cTP(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). II- 11
LexA- C - LexA E2F-1 - MBP MOCK
+ LexA sites
- LexA sites
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 32~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 32~- 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 24) and LexA (lane 6),but not with antisera against an u~elated 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 (26 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~-labeledyeast 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 BN1, 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. 38, 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~itro(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. II- L5
yTBP Lyzo MBP + -&a UV +
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~-labeledyeast 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 TR 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 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~-labeledyeast 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~itrotranscription 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 abili 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 % Activity rcj P, V, ri- 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 32~4abeledTFW, as well as TBP, as the target. As shown in Fig. 7A. LexA-E2F-1 yTBp-+ +++++ + 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~-labeledAd2ML 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 TFW (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 (yu)or TFW (yW) (3 pmol of each) as indicated. C, SDS-PAGE fractionation of UV- irradiated mixtures that contained 32~-labeledyeast 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~-labeledhuman TBP (3 prnol), photoreactive full-length LexA-E2F-1 (7.5 pmol), CYCl promoter DNA with or without two upstream Leu- 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 LeA-EX-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- - + + + + + yTBP++ - + + - - 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 Led, and R. Ebright and J. Greenblatt for helpful advice. REFERENCES 1. Zawel, L., and Reinberg, D. (1993) Prog. Nucleic Acids Res. Mol. Biol. 44, 67-108. 2- Hori, R., and Carey, M. (1994) Curr. Opin. Genet. Dev. 4, 236-244 3. Choy, B., and Green, M. R. (1993) Nature 366, 531-536 4. Stringer, K. F., Ingles, C. J., and Greenblatt, C. J. (1990) Nature 345, 783-786 5. Roberts, S. G., and Green, M. R. (1994) Nature 371, 717-720 6. Xiao, H., Pearson, A., Coulombe, B., T'ruant, R., Zhang, S., Regier, J. L., Triezenberg, S. J., Reinberg, D., Flores, O., Lngles, C. J., and Greenblatt, J. (1994) Mol. Cell. Biol. 14, 701 3-7024 7. Goodrich, J. A., Hoey, T., Thut, C. J., Admon, A., and Tjian, R. (1993) Ce11 75, 519-530 8. Ge, H., and Roeder, R. G. (1994) Cell78,513-523 9. Hoey, T., Weinzerl, R. O. J., Gill, G., Chen, J.-L., Dynlacht, B. D., and Tjian, R. (1993) Cell72, 247-260 10. Emili, A., Greenblatt, J., and Ingles, C. J. (1994) Mol. Cell. Biol. 14, 1582-1593 11. 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Chem. 267, 7845-7855 33. Ma, D., Watanabe, H., Mermelstein, F., Admon, A., Oguri, K., Sun, X., Wada, T., Imai, T., Shiroya, Reinberg, D., and Handa, H. (1993) Genes & Dev. 7, 2246-2257 34. Sun, X., Ma, D., Sheldon, M., Yeung, K., and Reinberg, D. (1994) Genes & Dev. 8, 2336-2348 35. Yokomori, K., Zeidler M. P., Cl-ien j.-L., Verrijzer C. P., Mlodzik M., and Tjian, R. (1994) Genes & Dev. 8, 2323-2323 36. Wang, W., Gralla, J. D., and Carey, M. (1992) Genes & Dev. 6, 1716-1727 37. Liljelund, P., Ingles, C. J., and Greenblatt, J. (1993) " Altered promoter binding of the TATA box-binding factor induced by the transcriptional activation domain of VP16 and suppressed by TFIIA" Mol. Gen. Genet. 241, 694-699. cmIII 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]) III- 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 characteristics 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 el~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 xtclA 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°/~-polyacry1amidegel. 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 AND DISCUSSION To study interactions between an 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 32~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 machinery as MBP-derivatized LexA-E2F-1 strongly activated transcription in a yeast extract enriched for components of the pol 11 transcriptional machinery (Fig. '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 III- 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). III- 11 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 Mr 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 at 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 ushg both a single copy integrated GAL4 responsive lncZ reporter gene (line 1) or each of two multicopy 1ncZ reporters Glucose 30°C Galactose Glucose 30°C 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 xtcld (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 (pADH1).d, Growth of isogenic wild type and xtcld 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 wtcU 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 wtclA 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 "~~uelchin~"~~(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 xklA 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 xtcld cells in the [II- 16 Activator Reporter p gai Units Table 1. Hyperactivation of transcription in XïCl 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 GALl 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 de~cribed~~. 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%. III- 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. III- 19 REFERENCES Conaway, R. C., & Conaway, J. W. Annri. Rev. Biochem. 62, 161-190 (1993). Struhl, K. Annu. Rex Genet. 29, 651-674 (1995). Ptashne, M. Nature 335, 683-689 (1988). Tjian, R., & Maniatis, T. Ce11 77, 5-8 (1994). Struhl, K. Ce11 84, 179-182 (1996). Emili, A., & Ingles, C. J. \. Biol. Clzern. 270, 13674-13680 (1995). Flemington, E. K., Speck, S. H., & Kack, W.C. Prx. IW!. Arc!! Sci. U.S.A. 90, 6914- 6918 (1993). Dorman, G., & Prestwich, G. D. Biochemistry 33, 5661-5673 (1994). Emery, H. S., Schild, D., Kellogg, D. E., & Motimer, R. K. Gene 104, 103-106 (1991). Gorbalenya, A. E., Koonin, E. V., Donchenko, A.P. & Blinov, V. M. Niicl. Acids Res. 17, 4713-4730 (1989). Ma, J., & Ptashne, M. Ce11 48, 847-853 (1987). Sadowski, I., Ma, J., Triezenberg, S., & Ptashne, M. Nnture 335, 563-564 (1988). Cress, W. D., & Triezenberg, S. J. Science 251, 87-90 (1991) Koleske, A. J., Buratowski, S., Nonet, M., & Young, R. A. Cc11 69, 583-894 (1992). Candau, R., & Berger, S. L. \. Biol. Chem. 271, 5237-5245 (1996). Neigebom, L., & Carlson, M. Genetics 108, 845-858 (1984). Balciunas, D., & Rome, R. Niicl. Acids Res. 23, 4421-4425 (1995). Yocum, R. R., Hanley, S., West, R., Ir., & Ptashne, M. Mol. Cell. Biol. 4, 1985-1998 (1984). Brent, R., & Ptashne, M. Ce21 43, 729-736 (1985). Allison, L. A., & ingles, C. J. Proc. Natl. Acnd. Sci. U.S.A. 86, 2794-2798 (1989). Gill, G., & Ptashne, M. Nature 334,721-724 (1988). Berger, S. L., Pina, B, Silverman, N., Marcus, G. A., Agapite, J., Regier, J. L., Triezenberg, S. J., & Guarente, L. Ce21 70,251-265 (1992). Yeung, K. C., Lnostroza, J. A., Mermelstein, F. H-, Kannabiran, C., & Reinberg, D. Genes Devi. 8, 2097-2109 (1994). Merino, A., Madden, K. R., Lane, W. S., Champoux, J. J., & Reinberg, D. Nntrlre 365,227-232 (1993). Auble, D. T., Hansen, K. E., Mueller, C. G. F., Lane, W. S., Thorner, J., & Hahn,S. Gerzes Devl. 8, 1920-1934 (1994). He, Z., Brinton, B. T., Greenblatt, J., Hassell, J. A., & ingles, C. J. Cd73, 1223-1232 (1993 Poon, D-, Campbell, A. M., Bai, Y., & Weil, P. A. \. Biol. Chem. 269, 23135-23140 (1994). Kim, Y.+, Bjorklund, S., Li, Y., Sayre, M. H., & Komberg, R. D. Cell 77, 599-608 (1994). Wang, R., Kobayashi, R., & Bishop, J. M. Proc. Nntl. Acnd. Sci. Lf.5.A.93, 8425-8430 (1996). 30. Gietz, D., St. Jean, A., Woods, R. A., & Schiestl, R. H. Nzicl. Acids Res. 20, 6 (1992). CHAPTER IV Interaction of the C-terminal Domain of the Largest Subunit of RNA Polyrnerase II with the Essential Splicing Factor PSF and the Putative Splicing Factor p54nrb This chapter represents preliminary work still in progress. (Except for the protein microsequencing, 1 did al1 of the experiments in this chapter tvith sorne technical assistance from M. Shales) IV- 1 SUMMARY 1 have used affinity chromatography to characterize human proteins which interact with the unique and essential C-terminal domain (Cm) 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 An 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 [y32~]~TP(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°/~-polyacrylamidegel 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 as 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 aytly Naht kr Pro 3.r 1 TyrkrProThrtrrPro*L. 2 Tyr Glu Pm lup kt Pro Cly Gfy 3 ~yrnar Pro bta Sor Pro kr 4 ~yrkrPro~$u?mâu S Tyr Sar Pm Thr kr Pm Sar 6 Tyr kr Pro Th kr Pm A8a 7 Tyr wr Pro Zhr kr Pm 3.r 6 Tyr kr 9m Th kt Pro 3.r 9 Tyr kr Pm Thr kr Pro kr 10 +yr *r Pro Zhr 3.r Pm âu Il Tyr kr Pm Zhr kr Pro Ilu 12 TyrsuProThrSuPmhr 13 fyt 5.r Pm Thr kr Oro kr 14 Tyr kr Pro ftu kr Pm kr 5 tyt wr 9m Zhr Su Pm 3.r t8 ~yrSar Oro Zhr kr Pm Su 17 tyr rrpro ~hrkr ?m âu 18 Tyr Smr Pm Zhr kr Pm 5.r 19 Tyr kr Pm Zhr kr Pro &r aI Tyr 5.r Pro Zhr trr Pro Sor 66 - n lyr SU sm ~hr5.r Pm kr P ryr kr Pro Thr kr Pm iua zl ryrsrrProibrkrProA8a CTD -, # syr tb.r pro rbr sr Pm %r a ryr sar Pm Zhr Smr Pm 3.r ryr ar Pm Thr kr Pm Am 27 TyrIhrPro?hzSmrPmA8~ fyr 5.r Pm Zhr kr Pm 3.r 29 Tyr Mr Pm Thr kr Pro Sa= JO Tyr Slr Pro Thr kr Pro Sor 3I Tyr kr Pro kr S.r: Pm Irg a ryr Pro GLO kr Pm % p Tyr Ihr Pro kr kr Pro Su 3 fyt Sor Pro kr kr Pm kr 35 Tyr ?L.r Pro kr Pm Ly8 S Tyr Tür Pm Zhr kr Pro 3.r 37 Zyr Slr Pro kr kr Pro Glu 38 Tyr Tür Pro Zhr 3.r Pm Ly8 1D Tyr 9ir Pm Thr kr Pro Lya Q Tyr kr Pro Thr kr Pm Ly8 41 Tyr 5.r Pm Zhr kr Pro Thr 4 Tyr Sor Pm nu Pro LYS 4 Tyr kr Pro n%r kr Pm Thr U Tyr Sar Pm Zhr 3U Pro Vai lb Tyr Thr Pro Thr kr Pm Lys Tyr wr Pm Zhr Smr Pm Thr 47 Tyr Sar Pm Zhr kr Pm Lyi 40 Tyr kr Pro Thr SuPm Sor Q Tyr ar Pm Zhr 3.r Pro Ly8 Cly $or Tk 9D Tyr kr Pm Zhr kr Pm Wy 5ï Tyr kr Pro n%r kr Pro Thr Coomassie Western Q Tyr Sar Lm Thr kr Pro Na rt.raRo4yp~4QnCLi~C(rrm stain 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 IV- 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. IV- 11 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 afhity 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 ~5Pbare highlighted by arrows. C and D, amino acid sequence of the hurnan PSF and p~4mbpolypeptides. 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 as 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 foms 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 foms of either PSF and p54*rb that were CO-expressedin bacteria and CO-purifiedas 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 IV- 16 El coli Exttact cnLA1 - % 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 p5Pb (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. IV- 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, it 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 p54~bcould 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, irl 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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Dong, B., Horowitz, D. S., Kobayashi, R., and Krainer, A. R. 1993. Purification and cDNA cloning of HeLa ce11 p54nrb, a nuclear protein with two RNA recognition motifs and extensive homology to human splicing factor PSF and Drosopli iln NANA/B J6. Nitc. Acids Res. 21:4085-4092. Emili, A., Greenblatt, J., and hgles, C. J. 1994. Species-specific interaction of the glutamine-rich activation domains of Spl with the TATA Box-binding portein. Mol. Cell. Biol. 14:1582-1593. Emili, A. and Ingles, C. J. 1995. Promoter-dependent Photo-Crosslinking of the Acidic Transcriptional Activator E2F-1 to the TATA-Binding Protein. /. Biol. Chem. 270:13674-13680. Gozani, O., Patton, j. G., and Reed, R. 1994. A novel set of spliceosome- associated proteins and the essential splicing factor PSF bind stably to pre- mRNA prior to catalytic step II of the splicing reaction. EMBO 1. 13:3356-3367. Green, M. R. 1991. Biochemical mechanisms of constitutive and regulated pre- mRNA sp licing. Aiznrl. Rrv. Cell Biol. 7559-599. Hallier, M., Tavitian, A., and Moreau-Cachelin, F. 1996. The transcription factor Spi/PU.l binds RNA and interferes with the RNA-binding protein p54nrb./. Bid. Chem. 271: 11177-1 1181. Huang, S., and Spector, D. L. 1996. intron-dependent recruitment of pre- mRNA splicing factors to sites of transcription. 1. Cr11 Biol. 133:719-732. Jimenez-Garcia, L. F., and Spector, D. L. 1993. In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism. Cell 73:47-59. Kang, M. E., and Dahmus, M. E. 1995. The photoactivated cross-linking of recombinant C-terminal domain to proteins in a HeLa ce11 transcription extract that comigrate with transcription factors IIE and IIF. 1. Biol. Cliem. 270:23390-23397. Kim Y.+, Bjorklund, S., Li, Y., Sayre, M.H., and Komberg, R.D. 1994. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cr11 77:599-608. Kim Y.-J., Bjorklund, S., Li, Y., Sayre, M.H., and Komberg, R.D. 1994. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cd 77:599-608. Lamm, G. M., and Lamond, A. 1. 1993. Non-çnRNP protein splicing factors. Biochi~ri. Biophys. Actn. ll73:247-265. Liao, S.-M., Zhang, J., Jeffery, D. A., Koleske, A. J., Thompson, C. M., Chao, D. M., Viljoen, M., van Vuuren, H. J. J., and Young, R. A. 1995. A kinase-cyclin pair in the RNA polymerase II holoenzyme. Nnttrre 374193-196. Matunis, E. L., Matunis, M. j., and Dreyfus, G. 1993. Association of individual hnRNP proteins and snRNPs with nascent transcripts. \. Ce11 Biol. 121:219-228. Maxon, M. E., Goodrich, J. A., and Tjian, R. 1994. Transcription factor IIE bkds preferentially to RNA polymerase IIa and recruits TFIIH: a mode1 for promoter clearance. Genes Dev. 8:515-524. McCracken, S., Fong, N., Yankulov, K., Ballantyne, S., Pan, G., Greenblatt, J., Patterson, S. D., Wickens, M., and Bentley, D. L. 1996. The C-terminal domain of RNA polymerase II couples mRNA processing to transcription: evidence for a "mRNA factory". Submitted for publication. Mortillaro, M. J., Blencowe, B. J., Wei, X., Nakayasu, H., Du, L., Warren, S. L., Sharp, P. A. and Berezney, R. 1996. A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix.Proc. Nd.Acnd. Sci. USA 93:8253-8257. Moyle, M., Lee, A., Anderson, W., and hgles, C. J. 1989. The C-terminal domain of the largest subunit of RNA polymerase II and transcription initiation. Mol. Cell. Biol. 9:5750-5753. Mullen, M. P., Smith, C. W. J., Patton, J. G., and Nadal-Ginard, B. 1991. a- tropornyosin mutually exclusive exon selection: cornpetition between branchpoint/polypyrimidine tracts determines default exon choice. Genes Dezt 5:642-655. Newman, A. J. 1994. Pre-mRNA splicing. Curr. Opin. Gefle. Deu. 4298-304. O'Keefe, R. T., Mayeda, A., Sadowski, C. L., Krainer, A. R., and Spector, D. L. 1994. Disruption of pre-mRNA splicing in vivo results in reorganization of splicing factors. \. Ce11 Biol. 124:249-260. Patterson, B., and Guthrie, C. 1991. A U-rich tract enhances usage of an alternative 3' splice site in yeast. Ce11 64:181-187. Patton, J. G., Porro, E. B., Galceran, J., Tempst, P., and Nadal-Ginard, B. 1993. Cloning and characterization of PSF, a novel pre-mRNA splicing factor. Gelles Dev. 7:393-406. Richler, C., Ast, G., Goitein, R., Wahrman, J., Sperling, R., and Sperling, J. 1994. Splicing components are excluded from the transcriptionally inactive XY body in male meiotic nuclei. Mol. Biol. Ce11 51341-1352. Rio, D. C. 1993. Splicing of pre-mRNA: mechanism, regulation and role in development. Clirr. Opin. Gene. Dez?. 3:574-584. Roscigno, R. F., Weiner, M., and Garcia-Blanco, M. A. 1993. A mutational analysis of the polypyrimidine tract of introns. [. Biol. Chem. 268-11222-11229. Shapiro, D. 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Pre-messenger RNA splicing of transcripts synthesized from human small nuclear RNA gene promoters. Bioche~il. Biophys. Res. Commun. 1951394-1400. Wuarin, J., and Schibler, U. 1994. Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing. Mol. Ccll. Biol. 14:7219-7225. Yang, Y.-S., Hanke, J. H., Caraannopoulos, L., Craft, C. M., Capra, J. D., and Tucker, P. W. 1993. Nono, a non-POU-domain-containing, octarner-binding protein, is the rnammalian homolog of Drosophila ~ZOIIA~~SS-Mol. Cdl. Biol. 2 3:5593-5603. Yuryev, A., Patturajan, M., Ltingtun, Y., Joshi, R. V., Gentile, C., Gebara, M., and Corden, J. 1996. The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins. Proc. Nnfl. Acd Sci. USA 936975-6980. Zhang, G., Taneja, K. L., Singer, R. H., and Green, M. R. 1994. Localization of pre-mRNA splicing in mammalian nuclei. Nntzire 3739309-812. APPENDIX The RNA Polymerase II C-Terminal Domain: Links to a Bigger and Better 'Holoenzyme'? Andrew Emili and C. James Ingles ïhis review was published in Current Opinion in Genetics and Development Vol 5, April 1995 SUMMARY The largest subunit of eukaryotic RNA polymerase Ii has an unusual tandemly repeated heptapeptide sequence at its carboxyl-terminus. The function of this evolutionarily conserved C-terminal domain is not known. New evidence, however, links it to the formation of a large multi- component RNA polymerase II complex possessing enhanced transcriptional initiation properties. The existance of a preassembled RNA polymerase II 'holoenzyme' in the ce11 calls into question the long held view of transcription initiation as an ordered promoter-dependent process. INTRODUCTION A major new character, the 'holoenzyme', has emerged in recent studies of RNA polymerase II (Pol II), the enzyme responsible for the synthesis of messenger RNA in eukaryotes. Holoenzyme is the name given to two related forms of Pol II in which the 12 subunit core enzyme is found associated with a number of accessory transcription factors [1@.,2a .]. Unlike the minimal core enzyme, which permits only a low basal level of transcription i~cz~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~itroand, 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. ml. 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 hlly 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 irl 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 SN1 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 TFW 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 appear to stimulate 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. An 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. REFERENCES 01. Koleske AJ, Young RA: An RNA polymerase II holoenzyme responsive to activators. Nntrire 1994, 368:466-469. A high Mr complex isolated from yeast cells was shown to contain RNA polymerase iI, SRB proteins and the general initiation factors TFIIB, TF[IF, and TFIIH. This preassembled 'holoenzyme' was responsive in vitro to the transcriptional activator GAL4-VP16. It may represent the fom of Pol II recruited to promoters in vivo. m.2. Kim Y-J, Bjorklund S, Li Y, Sayre MH, Komberg RD: A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Ce11 1994, 77:599-608. This paper describes a complex isolated from yeast cells, termed the 'mediator', which permits RNA polymerase II and its general initiation factors to respond to transcriptional activators in ~itro.Amongst its -20 polypeptides were TFIIF, GALII, SUGI, and the SRB proteins found in the form of holoenzyme described in ref 1. This mediator complex could also be isolated with the 12 core subunits of Pol Il as part of a holoenzyme with enhanced transcriptional properties. 3. Conaway RC, Conaway JW: General initiation factors for RNA polymerase II. 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This speculative review suggests that the repetitive heptapeptide domain of Pol II (the CTD) could bind proteins involved in hnRNA processing thereby facilitating the splicing of nascent pol II RNA transcripts and linking this process to RNA synthesis. IMAGE EVALUATION TEST TARGET (QA-3) APPLIED - IMAGE. lnc -= 1653 East Main Street ,--& ,--& Rochester. NY 14609 USA --= --= -- Phone: 71W82-0300 -1---- Fa: 716/288-5989 O 1993. Applied image. inc.. Ni Rights Reserved