1. Biochemistry and Chemical Biology
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Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

  1. Sarah K Stumper
  2. Harini Ravi
  3. Larry J Friedman
  4. Rachel Anne Mooney
  5. Ivan R Corrêa
  6. Anne Gershenson
  7. Robert Landick
  8. Jeff Gelles  Is a corresponding author
  1. Brandeis University, United States
  2. University of Wisconsin-Madison, United States
  3. New England Biolabs, Inc, United States
  4. University of Massachusetts, United States
Research Article
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Cite this article as: eLife 2019;8:e40576 doi: 10.7554/eLife.40576

Abstract

RNA polymerases (RNAPs) contain a conserved 'secondary channel' which binds regulatory factors that modulate transcription initiation. In Escherichia coli, the secondary channel factors (SCFs) GreB and DksA both repress ribosomal RNA (rRNA) transcription, but SCF loading and repression mechanisms are unclear. We observed in vitro fluorescently labeled GreB molecules binding to single RNAPs and initiation of individual transcripts from an rRNA promoter. GreB arrived and departed from promoters only in complex with RNAP. GreB did not alter initial RNAP-promoter binding but instead blocked a step after conformational rearrangement of the initial RNAP-promoter complex. Strikingly, GreB-RNAP complexes never initiated at an rRNA promoter; only RNAP molecules arriving at the promoter without bound GreB produced transcript. The data reveal that a model SCF functions by a 'delayed inhibition' mechanism and suggest that rRNA promoters are inhibited by GreB/DksA because their short-lived RNAP complexes do not allow sufficient time for SCFs to dissociate.

Data availability

All data analyzed for this study are included in the manuscript or in the source data files (doi:10.5281/zenodo.2530159).

The following data sets were generated

Article and author information

Author details

  1. Sarah K Stumper

    Department of Biochemistry, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Harini Ravi

    Department of Biochemistry, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Larry J Friedman

    Department of Biochemistry, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4946-8731
  4. Rachel Anne Mooney

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Ivan R Corrêa

    New England Biolabs, Inc, Ipswich, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Anne Gershenson

    Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Robert Landick

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5042-0383
  8. Jeff Gelles

    Department of Biochemistry, Brandeis University, Waltham, United States
    For correspondence
    gelles@brandeis.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7910-3421

Funding

National Institute of General Medical Sciences (R01GM081648)

  • Jeff Gelles

National Science Foundation (MCB-0446220)

  • Anne Gershenson

National Institute of General Medical Sciences (R01GM38660)

  • Robert Landick

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Antoine M van Oijen, University of Wollongong, Australia

Publication history

  1. Received: July 29, 2018
  2. Accepted: February 4, 2019
  3. Accepted Manuscript published: February 5, 2019 (version 1)
  4. Version of Record published: February 18, 2020 (version 2)

Copyright

© 2019, Stumper et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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