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 Our iUe NmrefBrBlIcB The author has granted a non- L'auteur a accorde une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/fïlm, de reproduction sur papier ou sur format électronique. 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