Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Structure and requirement of the Spt6 SH2 domain and an in vitro system to test the “torpedo model” of transcription termination Stefan Dengl aus München 2009 Erklärung II Erklärung Diese Dissertation wurde im Sinne von §13 Abs. 3 der Promotionsordnung vom 29. Januar 1998 von Herrn Prof. Dr. Patrick Cramer betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig und ohne unerlaubte Hilfe erarbeitet. München, am _______________________________ Stefan Dengl Dissertation eingereicht am 1. Gutachter: Prof. Dr. Patrick Cramer 2. Gutachter: Prof. Dr. Dietmar Martin Mündliche Prüfung am 25. März 2009 Acknowledgements III Acknowledgements First of all I would like to thank my supervisor Prof. Dr. Patrick Cramer for giving me the opportunity to work on challenging projects in a highly interdisciplinary manner. The freedom to realize own ideas in combination with your continuous advice and motivation is invaluable for the enthusiasm of a young scientist. I want to thank my collaborators Andreas Mayer, Mai Sun, Kristin Leike, Matthias Siebert and Johannes Soeding for their work and the discussions, but also for a lot fun and reflief from drawbacks in the projects. It was really a pleasure to work with you guys! Very special thanks go out to Claus Kuhn and Stephan Jellbauer, not only for teaching me crystallography and yeast work, respectively, but most important for your friendship in good and in hard times. The legendary "coffee-at-11.30" was often enough an escape from scientific misery and sometimes gave birth to new ideas. I will never forget the time we spent together, also outside of the lab. I wish to thank Elisabeth, Jasmin, Gerke and Florian for their help with elongation complexes. Laurent, Ale and Dirk for support concerning crystallography. Ania for advice on the ChIP-on- chip protocol. Claudia and Stefan Benkert for help with everything, without you the lab would be a battlefield. Many thanks to Heidi Feldmann for help with yeast work and for not killing me for some of my jokes. Thank you Gregor for illuminating me with your fluorescent expertise. I want to thank the former members of the lab, Hubert, Karim, Michela, Sabine, Sonja, Toni, and Tomislav for a lot of support especially in the beginnig. I want to thank you, Erika, for being an accomplice in "chromatin-related" issues that were otherwise quite exotic in the lab. Of course I want to thank everybody else in the Cramer group for help, media, buffers, DNA and protein standards, gels, coffee, cake, meaningful discussions and silly conversations. Von ganzem Herzen danke ich meinen Eltern und meiner Oma Elisabeth. Ohne eure moralische, aber auch finanzielle Unterstützung wäre das Studium und so viel mehr undenkbar gewesen. Dir Moni danke ich für das schöne letzte Jahr und dass du mir immer gute Laune verordnet hast auch wenn die Batterie mal leer war. Summary IV Summary During the biogenesis of messenger RNA, RNA polymerase II (RNAP II, Pol II) associates with numerous proteins and multiprotein complexes. These factors regulate the correct progression of the transcription cycle, which can be divided into three major phases: initiation, elongation and termination. This thesis describes the characterization of two important factors involved in two different phases of the transcription cycle in a highly interdisciplinary approach. Spt6 is an essential, modular protein involved in transcription elongation. It was characterized as a histone chaperone that binds to histones and assembles nucleosomes onto DNA after the passage of Pol II. In addition, it is linked to processes like splicing, mRNA processing and export, and histone modification. This functional versatility makes it a central player in the elongation process. The first part of this work shows the high resolution structure of the C-terminal SH2 domain of Spt6. The domain was shown previously to interact with the C-terminal domain (CTD) of Pol II, phosphorylated at Ser2 residues. Thus it links Spt6 functions directly to the transcription machinery. The domain has an unconventional binding specifity in contrast to the numerous SH2 domains involved in cellular signaling pathways. It binds phosphoserine instead of phosphotyrosine. In addition, it is the only SH2 domain encoded in the yeast genome, opposing a multitude of these domains in the genomes of higher eukaryotes. The X-ray structure gives insight into the peculiarities of this domain, with implications on its substrate specificity and molecular evolution. In this light, a model for the interaction with the CTD, as well as a deep analysis of the evolutionary relationship to other SH2 domains is presented. Microarray gene expression analysis shows the impact of a deletion of the SH2 domain on the transcription of the yeast genome. A genome wide localization map of Spt6, obtained by ChIP-on-chip experiments, is presented and compared to the localization of Pol II. The nuclear exoribonuclease complex Rat1/Rai1 plays a role in transcription termination. Two models explain the events at the end of a protein coding gene. The so-called „torpedo model“ states that Rat1/Rai1 processively degrades 3’ nascent RNA that is still attached to elongating Pol II after the mRNA product is cleaved by cleavage/polyadenylation factors. Upon contact with Pol II, Rat1/Rai1 is thought to disrupt the elongation complex and terminate transcription. There is however also data supporting the "allosteric model" that predicts the recruitment of a termination factor or the dissociation of an anti-termination factor. The second part of this thesis describes the establishment of a highly defined biochemical assay to test the "torpedo model" in vitro. A protocol for the expression and purification of the recombinant and active exonuclease complex and an additional interacting protein, Rtt103, was established. In combination with an improved in vitro elongation assay it is shown that Rat1 is not the dedicated termination factor that is predicted by the torpedo model. Publications V Publications Parts of this work have been published or are in the process of publication: Dengl S., Mayer, A., Sun, M., Cramer, P. (2009). Structure and widespread requirement of the Spt6 SH2 domain, the only SH2 domain in yeast. Journal of molecular biology, in revision. Dengl S., Cramer P. (2009). The torpedo nuclease Rat1 is insufficient to terminate RNA polymerase II in vitro. The Journal of biological chemistry, in revision. Sydow, J. F. , Brueckner, F., Dengl, S., Cramer P. (2009). Molecular basis of RNA polymerase II fidelity: mismatch specificity and RNA fraying. Manuscript in preparation. Cramer P, Armache KJ, Baumli S, Benkert S, Brueckner F, Buchen C, Damsma GE, Dengl S, Geiger SR, Jasiak AJ, Jawhari A, Jennebach S, Kamenski T, Kettenberger H, Kuhn CD, Lehmann E, Leike K, Sydow JF, Vannini A (2008) Structure of eukaryotic RNA polymerases. Annual review of biophysics 37: 337-352 Table of contents VI Table of contents Erklärung ....................................................................................................................II Ehrenwörtliche Versicherung ...................................................................................II Acknowledgements ..................................................................................................III Summary ...................................................................................................................IV Publications ...............................................................................................................V Table of contents .....................................................................................................VI 1 General introduction .............................................................................................1 1.1 Structure of RNA polymerase II and the elongation complex......................................1 1.2 The C-terminal domain (CTD) of Pol II subunit Rpb1....................................................3 1.3 The mRNA transcription cycle........................................................................................5 1.3.1 Transcription elongation ...................................... .......................................................6 1.3.2 Transcription termination ............................................................................................9 2 Materials and common methods .........................................................................12 2.1 Materials..........................................................................................................................12 2.1.1 Bacterial and yeast strains .......................................................................................12 2.1.2 Plasmids, oligonucleotides and peptides .................................................................12 2.1.3 Media and supplements ...........................................................................................15 2.1.4 Buffers and solutions ................................................................................................15 2.2 Common methods..........................................................................................................18 2.2.1 Molecular cloning .....................................................................................................19 2.2.2 Preparation of competent cells .................................................................................20