The Pennsylvania State University The Graduate School Eberly College of Science REGULATION OF TRANSCRIPTION BY NusA AND NusG IN Bacillus subtilis A Dissertation in Biochemistry, Microbiology, and Molecular Biology by Smarajit Mondal © 2016 Smarajit Mondal Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2016 The dissertation of Smarajit Mondal was reviewed and approved* by the following: Paul Babitzke Professor Biochemistry and Molecular Biology Dissertation Adviser Chair of Committee David S. Gilmour Professor of Biochemistry and Molecular Biology Joseph C. Reese Professor of Biochemistry and Molecular Biology Katsuhiko Murakami Professor of Biochemistry and Molecular Biology Philip C. Bevilacqua Professor Chemistry Scott B. Selleck Head of the Department of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School. ii ABSTRACT Transcription in bacteria is regulated at the level of initiation, elongation and termination. Although the regulation of transcriptional initiation is well studied, the regulation of elongation and termination are not well understood. This thesis focuses on understanding the role of NusA on intrinsic termination and the role of NusG on RNA polymerase pausing using genomic, biochemical and computational analyses. Tight regulation of transcription termination is required to maintain proper levels of gene expression in bacteria, because termination failure abolishes operon boundaries, leading to misregulation of downstream genes. NusA is a negative transcription elongation factor that was known to cause a slight stimulation of termination at intrinsic terminators in vitro, but its impact on termination and global gene expression in vivo was not known. In this thesis, I describe the mapping of intrinsic terminators genome wide in B subtilis and measure the effect of NusA on the efficiency of these terminators in vivo using a novel high resolution 3’ end-mapping technique coupled with mRNA profiling. Based on these studies, I report the existence of a subclass of previously unidentified pseudo-intrinsic terminators that are dependent on NusA for termination. Sequence comparison of different terminators reveals that weak hairpins and/or distal U-tract interruptions favors NusA-dependent termination, supporting a model in which NusA assists hairpin folding and slows down RNA polymerase near the termination window. These studies also revealed that readthrough of NusA-dependent terminators increases transcription of genes related to replication and DNA metabolism, suggesting a role of NusA in maintaining genome stability. I further show that nusA is autoregulated by a transcription attenuation mechanism that does not rely on antiterminator structures to prevent termination. Instead, NusA-stimulated termination in its 5'UTR dictates the extent of transcription into the operon. iii Another major focus of this thesis is to understand the regulation of transcription elongation by NusG-stimulated pausing of RNA polymerase. NusG is a positive elongation factor in E. coli that accelerates transcription by reducing the dwell time of RNA polymerase at pause sites. In B. subtilis, NusG stimulates pausing at positions U107 and U144 in the trp-leader transcript. NusG-stimulated pausing at U144 requires a short sequence in the non-template DNA strand and participates in the TRAP-dependent translation repression mechanism. In this thesis, I report the characterization of the NusG-stimulated U107 pause signal and show that disruption of the NusG recognition motif dramatically reduces pausing. These results suggest a mechanism in which RNA polymerase pausing at this site participates in the transcription attenuation mechanism by increasing additional time for TRAP binding to the nascent transcript. iv TABLE OF CONTENTS LIST OF FIGURE…………………………………………………………………...vii LIST OF TABLES…………………………………………………………………..viii ABBREVIATIONS………………………………………………………………….ix ACKNOWLEDGEMENTS………………………………………………………….x DEDICATION……………………………………………………………….………xi Chapter 1: Introduction: An overview of transctiption in bacteria ................................. 1 1.1 Components involved in prokaryotic transcription .................................................... 1 1.1.1 The RNA polymerase core enzyme ................................................................ 1 1.1.2 The sigma factor and RNAP holoenzyme ....................................................... 5 1.1.3 Other Regulatory factors ................................................................................. 7 1.1.3.1 Factors interacting with the secondary channel ................................. 7 1.1.3.2 NusA: a conserved termination and pausing factor ........................... 11 1.1.3.3 NusG: a cofactor of Rho-dependent termination ............................... 14 1.2 The transcription cycle ............................................................................................... 18 1.2.1 Promoter recognition and initiation ............................................................... 18 1.2.2 Promoter clearence and elongation ................................................................ 21 1.2.3 Termination and dissociation of individual components ............................... 22 1.2.3.1 Intrinsic termination ........................................................................... 22 1.2.3.2 Rho-dependent termination ................................................................ 25 1.2.3.3 Importance of termination .................................................................. 26 1.3 Transcriptional pausing .............................................................................................. 28 1.4 Transcriptional attenuation ........................................................................................ 32 1.4.1 Riboswitch-mediated attenuation ................................................................... 32 1.4.2 T-box antitermination ..................................................................................... 34 1.4.3 Attenuation by ribosomal stalling ................................................................... 34 1.4.4 Attenuation by RNA-binding proteins ........................................................... 35 1.5 References .................................................................................................................. 37 Chapter 2: NusA-dependent Transcription Termination Prevents Misregulation of Global Gene Expression ................................................................................................ 57 2.1 Abstract ...................................................................................................................... 58 2.2 Introduction ................................................................................................................ 59 2.3 Results ........................................................................................................................ 60 2.3.1 Construction of a NusA depletion strain ......................................................... 60 2.3.2 Identification of terminators by 3’ end-mapping ............................................ 61 2.3.3 Identification of NusA-dependent termination in vivo .................................... 66 2.3.4 Terminator features that affect their response to NusA .................................. 69 2.3.5 Weak terminators favor NusA-dependent termination ................................... 73 2.3.6 Examination of NusA-inhibited terminators in vitro ...................................... 75 2.3.7 NusA depletion leads to global gene expression defects ................................ 75 2.3.8 Misregulation of replication and DNA metabolism genes .............................. 77 2.3.8. Autoregulation of nusA expression ................................................................ 79 v 2.4 Discussion .................................................................................................................. 82 2.5 Materials and Methds ................................................................................................. 85 2.5.1 Library preparation .......................................................................................... 85 2.5.2 Data analysis ................................................................................................... 86 2.5.2.1 Identification of 3’ ends ..................................................................... 87 2.5.2.2 Terminator screening and characterization ........................................ 87 2.5.2.3 Calculation of termination efficiency ................................................. 88 2.5.2.4 Differential expression and GO-term analysis ................................... 89 2.5.3 Primer extension .............................................................................................. 90 2.5.4 Western blot .................................................................................................... 90 2.5.5 DNA templates, plasmids and proteins ........................................................... 90 2.5.6 In vitro termination ......................................................................................... 91 2.5.5 β-galactosidase assay ...................................................................................... 91 2.6 References .................................................................................................................. 92 Chapter 3: Characterization