1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Structural basis of transcription arrest by coliphage HK022 nun in an Escherichia coli RNA polymerase elongation complex

  1. Jin Young Kang
  2. Paul Dominic B Olinares
  3. James Chen
  4. Elizabeth A Campbell
  5. Arkady Mustaev
  6. Brian T Chait
  7. Max E Gottesman
  8. Seth A Darst  Is a corresponding author
  1. The Rockefeller University, United States
  2. Public Health Research Institute, United States
  3. Columbia University Medical Center, United States
Research Article
  • Cited 59
  • Views 2,938
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Cite this article as: eLife 2017;6:e25478 doi: 10.7554/eLife.25478

Abstract

Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λΔNA.To provide a structural framework to understand how Nun blocks RNAP translocation, we determined structures of Escherichia coli RNAP ternary elongation complexes (TECs) with and without Nun by single-particle cryo-electron microscopy. Nun fits tightly into the TEC by taking advantage of gaps between the RNAP and the nucleic acids. The C-terminal segment of Nun interacts with the RNAP β and β’ subunits inside the RNAP active site cleft as well as with nearly every element of the nucleic-acid scaffold, essentially crosslinking the RNAP and the nucleic acids to prevent translocation, a mechanism supported by the effects of Nun amino acid substitutions. The nature of Nun interactions inside the RNAP active site cleft suggests that RNAP clamp opening is required for Nun to establish its interactions, explaining why Nun acts on paused TECs.

Data availability

The following data sets were generated
    1. Jin Young Kang
    2. Seth A. Darst
    (2017) CryoEM structure of HK022 Nun - E. coli RNA polymerase elongation complex
    Publicly available at the EMBL-EBI Protein Data Bank in Europe (accession no: EMD-8584).
    1. Jin Young Kang
    2. Seth A. Darst
    (2017) CryoEM structure of crosslinked E.coli RNA polymerase elongation complex
    Publicly available at the EMBL-EBI Protein Data Bank in Europe (accession no: EMD-8585).
    1. Jin Young Kang
    2. Seth A. Darst
    (2017) CryoEM structure of E.coli RNA polymerase elongation complex
    Publicly available at the EMBL-EBI Protein Data Bank in Europe (accession no: EMD-8586).
The following previously published data sets were used
    1. Bae B
    2. Darst SA
    (2013) Crystal Structure Analysis of the E.coli holoenzyme
    Publicly available at the RCSB Protein Data Bank (accession no: 4LJZ).

Article and author information

Author details

  1. Jin Young Kang

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Paul Dominic B Olinares

    Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. James Chen

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Elizabeth A Campbell

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Arkady Mustaev

    Public Health Research Institute, Newark, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Brian T Chait

    Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Max E Gottesman

    Department of Microbiology and Immunology, Columbia University Medical Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Seth A Darst

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    For correspondence
    darst@rockefeller.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8241-3153

Funding

National Institutes of Health (R35 GM118130)

  • Seth A Darst

National Institutes of Health (R01 GM037219)

  • Max E Gottesman

National Institutes of Health (P41 GM103314)

  • Brian T Chait

Public Health Research Institute Research Support grant

  • Arkady Mustaev

National Institutes of Health (P41 GM109824)

  • Brian T Chait

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

Reviewing Editor

  1. Nikolaus Grigorieff, Janelia Research Campus, Howard Hughes Medical Institute, United States

Publication history

  1. Received: January 26, 2017
  2. Accepted: March 19, 2017
  3. Accepted Manuscript published: March 20, 2017 (version 1)
  4. Version of Record published: April 10, 2017 (version 2)

Copyright

© 2017, Kang 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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