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      Modular Organization of the NusA- and NusG-Stimulated RNA Polymerase Pause Signal That Participates in the Bacillus subtilis trp Operon Attenuation Mechanism

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          ABSTRACT

          The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to the nascent transcript and blocks the formation of an antiterminator structure such that the formation of an overlapping intrinsic terminator causes termination in the 5′ untranslated region (5′ UTR). In the absence of bound TRAP, the antiterminator forms and transcription continues into the trp genes. RNA polymerase pauses at positions U107 and U144 in the 5′ UTR. The general transcription elongation factors NusA and NusG stimulate pausing at both positions. NusG-stimulated pausing at U144 requires sequence-specific contacts with a T tract in the nontemplate DNA (ntDNA) strand within the paused transcription bubble. Pausing at U144 participates in a trpE translation repression mechanism. Since U107 just precedes the critical overlap between the antiterminator and terminator structures, pausing at this position is thought to participate in attenuation. Here we carried out in vitro pausing and termination experiments to identify components of the U107 pause signal and to determine whether pausing affects the termination efficiency in the 5′ UTR. We determined that the U107 and U144 pause signals are organized in a modular fashion containing distinct RNA hairpin, U-tract, and T-tract components. NusA-stimulated pausing was affected by hairpin strength and the U-tract sequence, whereas NusG-stimulated pausing was affected by hairpin strength and the T-tract sequence. We also determined that pausing at U107 results in increased TRAP-dependent termination in the 5′ UTR, implying that NusA- and NusG-stimulated pausing participates in the trp operon attenuation mechanism by providing additional time for TRAP binding.

          IMPORTANCE The expression of several bacterial operons is controlled by regulated termination in the 5′ untranslated region (5′ UTR). Transcription attenuation is defined as situations in which the binding of a regulatory molecule promotes transcription termination in the 5′ UTR, with the default being transcription readthrough into the downstream genes. RNA polymerase pausing is thought to participate in several attenuation mechanisms by synchronizing the position of RNA polymerase with RNA folding and/or regulatory factor binding, although this has only been shown in a few instances. We found that NusA- and NusG-stimulated pausing participates in the attenuation mechanism controlling the expression of the Bacillus subtilis trp operon by increasing the TRAP-dependent termination efficiency. The pause signal is organized in a modular fashion containing RNA hairpin, U-tract, and T-tract components.

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          Author and article information

          Contributors
          Role: Editor
          Journal
          J Bacteriol
          J. Bacteriol
          jb
          jb
          JB
          Journal of Bacteriology
          American Society for Microbiology (1752 N St., N.W., Washington, DC )
          0021-9193
          1098-5530
          15 May 2017
          27 June 2017
          15 July 2017
          : 199
          : 14
          : e00223-17
          Affiliations
          Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA
          University of Wisconsin—Madison
          Author notes
          Address correspondence to Paul Babitzke, pxb28@ 123456psu.edu .
          [*]

          Present address: Smarajit Mondal, Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA.

          S.M. and A.V.Y. contributed equally to this work.

          Citation Mondal S, Yakhnin AV, Babitzke P. 2017. Modular organization of the NusA- and NusG-stimulated RNA polymerase pause signal that participates in the Bacillus subtilis trp operon attenuation mechanism. J Bacteriol 199:e00223-17. https://doi.org/10.1128/JB.00223-17.

          Author information
          http://orcid.org/0000-0003-2481-1062
          Article
          PMC5494738 PMC5494738 5494738 00223-17
          10.1128/JB.00223-17
          5494738
          28507243
          b01df9b6-92a2-44dc-8a3d-6a9343c0b8a3
          Copyright © 2017 American Society for Microbiology.

          All Rights Reserved.

          History
          : 23 March 2017
          : 5 May 2017
          Page count
          Figures: 5, Tables: 0, Equations: 0, References: 50, Pages: 12, Words: 7522
          Funding
          Funded by: HHS | National Institutes of Health (NIH) https://doi.org/10.13039/100000002
          Award ID: GM098399
          Award Recipient : Paul Babitzke
          Categories
          Research Article
          Custom metadata
          July 2017

          NusA,transcription factors,transcription attenuation,gene regulation,TRAP,RNA polymerase pausing,NusG

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