1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation

  1. James Chen
  2. Saumya Gopalkrishnan
  3. Courtney Chiu
  4. Albert Y Chen
  5. Elizabeth A Campbell
  6. Richard L Gourse
  7. Wilma Ross
  8. Seth A Darst  Is a corresponding author
  1. The Rockefeller University, United States
  2. University of Wisconsin-Madison, United States
https://doi.org/10.7554/eLife.49375
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  1. James Chen
  2. Saumya Gopalkrishnan
  3. Courtney Chiu
  4. Albert Y Chen
  5. Elizabeth A Campbell
  6. Richard L Gourse
  7. Wilma Ross
  8. Seth A Darst
(2019)
E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation
eLife 8:e49375.
https://doi.org/10.7554/eLife.49375
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Abstract

TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp, but the structural basis for regulation by these factors remains unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis revealed TraR-induced changes in RNAP conformational heterogeneity. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

Data availability

The cryo-EM density maps have been deposited in the EMDataBank under accession codes EMD-0348 [Eco TraR-Eσ70(I)], EMD-0349 [Eco TraR-Eσ70(II)], EMD-20231 [Eco TraR-Eσ70(III)], EMD-20230 (Eco Eσ70), EMD-20203 (rpsT P2-RPo), and EMD-20232 (rpsT P2-RPo2). The atomic coordinates have been deposited in the Protein Data Bank under accession codes 6N57 [Eco TraR-Eσ70(I)], 6N58 [Eco TraR-Eσ70(II)], 6P1K (Eco Eσ70), and 6OUL (rpsT P2-RPo).

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Author details

  1. James Chen

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2311-003X

  2. Saumya Gopalkrishnan

    Department of Bacteriology, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Courtney Chiu

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

  4. Albert Y Chen

    Department of Bacteriology, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Elizabeth A Campbell

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

  6. Richard L Gourse

    Department of Bacteriology, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Wilma Ross

    Department of Bacteriology, University of Wisconsin-Madison, Madison, 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 (R01 GM114450)

  • Elizabeth A Campbell

National Institutes of Health (R01 GM37048)

  • Richard L Gourse

National Institutes of Health (R35 GM118130)

  • Seth A Darst

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

Copyright

© 2019, Chen 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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  1. James Chen
  2. Saumya Gopalkrishnan
  3. Courtney Chiu
  4. Albert Y Chen
  5. Elizabeth A Campbell
  6. Richard L Gourse
  7. Wilma Ross
  8. Seth A Darst
(2019)
E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation
eLife 8:e49375.
https://doi.org/10.7554/eLife.49375

Share this article

https://doi.org/10.7554/eLife.49375

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