Mechanism of Antitermination by NusG-like Proteins and the Role of RNAP Conformational Mobility in Transcription Cycle Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Anastasia Sevostiyanova, M.S. Microbiology Graduate Program The Ohio State University 2010 Dissertation Committee: Dr. Irina Artsimovitch, Advisor Dr. Michael Ibba Dr. Kurt Fredrick Dr. Mark Foster Copyright by Anastasia Sevostiyanova 2010 Abstract Uninterrupted synthesis of complete, up to a million nucleotides long, RNA chains by multi-subunit RNA polymerases (RNAPs) requires accessory proteins that help RNAP bypass numerous roadblocks it encounters along the way. Antitermination factors are found in all organisms from bacteriophages to humans and share the ability to switch the elongating RNAP into a highly processive state. Their molecular mechanisms, and in most cases even their binding sites on the transcription elongation complex (TEC), remain unknown. Diverse elongation factors, including bacteriophage λ N and Q proteins, HIV Tat, and regulators from the NusG family, help RNAP to bypass various pause and termination signals, thereby increasing its processivity. Bacterial factor RfaH, an operon-specific paralog of the general transcription factor NusG, is an excellent model for studies of the antitermination mechanism. RfaH acts as a canonical antiterminator: it increases the apparent elongation rate in vitro, reduces pausing, and facilitates bypass of some terminators. Studies of RfaH have already provided many insights into its function: we have obtained the RfaH structure and identified its binding sites on RNAP and the non- template (NT) DNA, identified many RfaH-controlled genes, and elucidated some ii aspects of the RfaH mechanism. I aimed to dissect the molecular contacts in the RfaH- modified TEC and to study the cellular context of RfaH action using a complex of in vivo and in vitro techniques. We demonstrated that RfaH increases the apparent elongation rate by preventing the TEC isomerization into an off-pathway state. Since RfaH binds 75Å away from the RNAP active site, where the structural rearrangements accompanying this isomerization occur, we envisioned that RfaH may transmit an allosteric signal to the active site. The complex and dynamic architecture of RNAP allows for many conformational changes whose regulatory importance we have just started to explore, and several hypothetical allosteric pathways have been proposed. In particular, five elements in the β‘ subunit, switch-2 (SW2), clamp, F-bridge helix and trigger loop (TL), appear to adopt different conformations in crystals and in solution. RfaH binds to the clamp, which is in turn directly linked to the SW2. Our data suggest that folding of the SW2 is crucial for transition from a catalytically-incompetent intermediate to the open promoter complex, and we are pursuing its role in elongation and termination. Our recent data led us to propose that that RfaH binds simultaneously to the β and β’ regions that constitute the clamp, thereby locking it in a closed conformation in which RNAP tightly encircles the nucleic acid chains. This closed conformation likely corresponds to the processive, pause- resistant state of the TEC. Presented work is focused on the mechanism of the NusG-like proteins and the conformational mobility of regulatory elements in RNAP that may play important roles throughout the transcription cycle. iii Acknowledgements First and most of all, I would like to thank my advisor Irina for creating an excellent working environment, high professional standards and the degree of scientific freedom she offered me, for her leadership, dedication to science, friendship, support, patience, driving lessons and my beloved cat. I also want to thank my Committee members, Dr. Kurt Fredrick, Dr. Mark Foster and Dr. Mike Ibba for their valuable feedback and support over the years. I thank the organizers of the memorable Mountain Lake meeting in 2008 for creating an intellectually stimulating environment and support. I want to express my gratitude to everyone who tried to control my life and failed. I thank Andrey Feklistov, Agus Muñoz Garcia, Amit Dashottar, Daniel Alpern and Olga Karicheva for their support, long talks and midnight walks. I want to thank Ran Furman, Noah Reynolds and Kiley Dare for their friendship and support. I want to thank my father for his support and encouragement to advance my career. iv This work is dedicated to the memory of my mother Elena. v Vita 2000-2005: College of Biological Sciences, Moscow State University, Moscow, Russia. 2005-2006: Junior Research Scientist, Laboratory of Molecular Genetics of Microorganisms, Institute of Molecular Genetics, Moscow, Russia 2006 to date: Department of Microbiology, Ohio State University, Columbus, OH Publications • Sevostyanova A and Artsimovitch I (2010) Nucleic Acids Res. Jul 17, PMID: 20639538 Functional analysis of Thermus thermophilus transcription factor NusG. • Pupov D, Miropolskaya N, Sevostyanova A, Bass I, Artsimovitch I & Kulbachinskiy A (2010) Nucleic Acids Res. May 10, PMID: 2045775 Multiple roles of the RNA polymerase β′ SW2 region in transcription initiation, promoter escape, and RNA elongation. • Belogurov GA, Sevostyanova A, Svetlov V, Artsimovitch I (2010) Mol Microbiol 76(2), 286-301. Functional regions of the N-terminal domain of the antiterminator RfaH. • Belogurov GA, Vassylyeva MN, Sevostyanova A, Appleman JR, Xiang AX, Lira R, Webber SE, Klyuyev S, Nudler E, Artsimovitch I & Vassylyev DG. (2009) Nature 457(7227), 332-35. Transcription inactivation through local refolding of the RNA polymerase structure. vi • Sevostyanova A, Svetlov V, Vassylyev D G & Artsimovitch I (2008) PNAS 105, 865-70. The elongation factor RfaH and the initiation factor sigma bind to the same site on the transcription elongation complex. • Sevostyanova A, Feklistov A, Barinova N, Heyduk E, Bass I, Klimasauskas S, Heyduk T & Kulbachinskiy A (2007) J Biol Chem 282, 22033-9. Specific recognition of the -10 promoter element by the free RNA polymerase sigma subunit. • Sevostyanova A, Djordjevic M, Kuznedelov K, Naryshkina T, Gelfand MS, Severinov K.& Minakhin L.(2006) J Mol Biol 366, 420-35. Temporal regulation of viral transcription during development of T. thermophilus bacteriophage ϕYS40. • Feklistov A., Barinova N., Sevostyanova A., Heyduk E., Bass I., Vvedenskaya I., Kuznedelov K., Merkiene E., Stavrovskaya E., Klimasauskas S., Nikiforov V., Heyduk T., Severinov K. & Kulbachinskiy A. (2006) Mol Cell 23, 97-107. A basal promoter element recognized by free RNA polymerase sigma subunit determines promoter recognition by RNA polymerase holoenzyme. Fields of study Major Field: Microbiology vii Table of Contents Abstract ......................................................................................................................................... ii Acknowledgements ................................................................................................................... iv Vita ................................................................................................................................................ vi Publications ................................................................................................................................. vi Fields of study ........................................................................................................................... vii Table of Contents ..................................................................................................................... viii List of Figures ........................................................................................................................... xiii List of Tables ............................................................................................................................ xvii List of Symbols and Abbreviations .................................................................................... xviii Chapter 1: Introduction ............................................................................................................... 1 Basics of Transcription ................................................................................................................. 1 RNA Polymerase Architecture ................................................................................................... 1 Transcription Cycle .................................................................................................................... 4 Nucleotide Addition Cycle.......................................................................................................... 6 Transcription Initiation .............................................................................................................. 12 Initial Promoter Recognition .................................................................................................... 12 Intermediates in Open Complex Formation ............................................................................. 13 Transition to Elongation .......................................................................................................... 16 Transcription Pausing and Termination .................................................................................. 18 Role of Pausing in Transcription Regulation ........................................................................... 18 viii Cellular