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Transcriptional Termination by Bacterial RNA Polymerase

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GENOME-SCALE STUDIES OF RHO-DEPENDENT TRANSCRIPTION TERMINATION IN ESCHERICHIA COLI by Jason Matthew Peters A dissertation submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy (Genetics) at the UNIVERSITY OF WISCONSIN – MADISON 2012 Date of final oral examination: 8/10/12 The dissertation is approved by the following members of the Final Oral Committee: Robert Landick, Professor, Biochemistry Gary Roberts, Professor Emeritus, Bacteriology Richard Burgess, Professor Emeritus, Oncology Audrey Gasch, Associate Professor, Genetics Nicole Perna, Associate Professor, Genetics i Genome-scale studies of Rho-dependent transcription termination in Escherichia coli Jason Matthew Peters under the supervision of Professor Robert Landick University of Wisconsin – Madison Rho is a homohexameric ring-shaped protein that translocates nascent RNA through its central cleft in an ATP-dependent manner, then dissociates RNA polymerase (RNAP) from RNA and template DNA. Despite decades of study, little was known about Rho association with transcription elongation complexes (ECs); sites of Rho termination across the E. coli chromosome; and the effects of the elongation factors NusG, NusA, and the nucleoid structuring protein H-NS on Rho termination. I found that Rho and RNAP have very similar distributions on DNA, suggesting that Rho associates with ECs early and throughout the process of transcription elongation. This association allows Rho to quickly respond to termination signals such as those that are unmasked when transcription and translation become uncoupled. Prior to my studies, the sites of Rho termination in E. coli were mostly unknown. I identified Rho-dependent terminators by examining the distribution of RNAP on DNA in Rho-inhibited cells. I found that Rho terminates highly structured RNAs, such as transfer RNAs (tRNAs) and small RNAs (sRNAs). This finding was surprising, because it was ii thought that Rho could not associate with structured RNAs. I also found that Rho terminated a small set of novel antisense transcripts that occurred within genes. Upon examining the transcriptome of cell in which Rho was inhibited, I determined that widespread increases in antisense transcription occurred throughout the genome. This finding established that a major function of Rho in vivo is to prevent elongation of antisense transcripts. Finally, I investigated the effects of NusG, NusA, and H-NS on Rho termination. I found that NusG enhances termination at a minority subset of Rho-dependent terminators that are defined by a low C/G ratio at the termination site. In contrast, a large deletion within nusA had no effect on Rho termination in vivo. I also identified a strong overlap between Rho-dependent terminators and H-NS binding sites, as well as genetic interactions between hns and rho. This led me to propose a model in which H- NS enhances Rho termination by slowing RNAP elongation, providing a longer kinetic window for Rho to terminate transcription. iii Acknowledgements I give my thanks to Bob, Rachel, and members of the Landick and Gourse labs for their patience in enduring more than a half a decade worth of bad jokes. I would also like to give special thanks to the transcription termination factor Rho. I couldn’t have done it without you, buddy. iv Table of contents Abstract ........................................................................................................................... i Acknowledgements ....................................................................................................... iii Table of contents...........................................................................................................iv List of figures .............................................................................................................. xiii List of tables .............................................................................................................. xviii Chapter 1 - Introduction ................................................................................................ 1 Introduction to transcription termination ....................................................................... 2 Elongation complex stability and the definition of termination ...................................... 3 The intrinsic termination signal and steps in the pathway of intrinsic termination ........ 7 The intrinsic termination signal ................................................................................. 7 Pausing at the intrinsic terminator ............................................................................ 8 Terminator hairpin nucleation ................................................................................. 11 Possible role of the partially formed terminator hairpin in pausing ......................... 12 Commitment to the termination pathway ................................................................ 15 Sequence conservation of intrinsic terminators ......................................................... 16 The mechanism of intrinsic termination ..................................................................... 18 v Changes in the nucleic acid scaffold during intrinsic termination ........................... 19 Conformational changes in RNAP during intrinsic termination ............................... 20 Intrinsic termination in other bacteria ......................................................................... 23 Introduction to Rho-dependent termination ................................................................ 25 The Rho termination signal and steps in the pathway of Rho termination ................. 26 Rho binding to RNA ............................................................................................... 26 RNA translocation through Rho.............................................................................. 32 EC pausing at the site of termination ..................................................................... 33 Kinetic coupling ...................................................................................................... 34 Rho association with elongation complexes .............................................................. 35 The mechanism of Rho termination ........................................................................... 36 Changes in the nucleic-acid scaffold during Rho termination ................................. 37 Conformational changes in RNAP during Rho Termination ................................... 39 Physiological functions and targets of Rho ................................................................ 40 Targets of Rho ....................................................................................................... 40 Silencing of foreign DNA ........................................................................................ 41 Suppression of antisense transcription .................................................................. 42 vi The mechanism of Rho-dependent polarity ............................................................... 42 Rho-ribosome competition for RNA ........................................................................ 43 Rho-ribosome competition for NusG ...................................................................... 43 Two-checkpoint model for polarity.......................................................................... 47 Protein factors that may affect Rho termination ......................................................... 47 NusA ...................................................................................................................... 47 H-NS ...................................................................................................................... 48 Conclusions ............................................................................................................... 50 Outline of thesis chapters .......................................................................................... 51 References ................................................................................................................ 53 Chapter 2 - Rho trafficking on bacterial transcription units in vivo........................ 69 Abstract ..................................................................................................................... 70 Introduction ................................................................................................................ 71 Results ....................................................................................................................... 76 Analysis of RNAP ChIP-chip signals on E. coli TUs ............................................... 76 Regulator trafficking on representative E. coli TUs ................................................ 77 σ70, NusA, NusG, and Rho associate with ECs in different patterns ...................... 80 vii NusG apparent occupancy depends on TU length, not gene function ................... 89 Promoter-proximal RNAP peaks correlate with promoter-proximal NusA and Rho peaks ..................................................................................................................... 92 Rho-dependent termination is not the primary cause of promoter-proximal RNAP peaks ..................................................................................................................... 96 Discussion ................................................................................................................
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