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Structural origins of Escherichia coli RNA polymerase open promoter complex stability

View ORCID ProfileRuth M. Saecker, View ORCID ProfileJames Chen, Courtney E. Chiu, View ORCID ProfileBrandon Malone, Johanna Sotiris, Mark Ebrahim, View ORCID ProfileLaura Y. Yen, View ORCID ProfileEdward T. Eng, View ORCID ProfileSeth A. Darst
doi: https://doi.org/10.1101/2021.09.08.459427
Ruth M. Saecker
aLaboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065 USA
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  • ORCID record for Ruth M. Saecker
  • For correspondence: rsaecker@rockefeller.edu darst@rockefeller.edu
James Chen
aLaboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065 USA
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Courtney E. Chiu
aLaboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065 USA
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Brandon Malone
aLaboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065 USA
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Johanna Sotiris
bThe Evelyn Gruss Lipper Cryo-Electron Microscopy Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065 USA
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Mark Ebrahim
bThe Evelyn Gruss Lipper Cryo-Electron Microscopy Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065 USA
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Laura Y. Yen
cThe National Resource of Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027 USA
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Edward T. Eng
cThe National Resource of Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027 USA
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Seth A. Darst
aLaboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065 USA
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  • ORCID record for Seth A. Darst
  • For correspondence: rsaecker@rockefeller.edu darst@rockefeller.edu
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Abstract

The first step of gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms “open” complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo but their structural origins remain largely unknown. Here we use cryo-electron microscopy to determine structures of RPo formed de novo at three promoters with widely differing lifetimes at 37°C: λPR (t1/2 ∼ 10 hours), T7A1 (t1/2 ∼ 4 minutes), and a point mutant in λPR (λPR-5C) (t1/2 ∼ 2 hours). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate-strand “scrunches” inside the active site cleft; the template-strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate RPo lifetime and affect the subsequent steps of the transcription cycle.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Author Contributions: Conceptualization, R.M.S. and S.A.D.; Methodology, R.M.S., J.C., C.E.C. and S.A.D.; Investigation, R.M.S., J.C., C.E.C, B.M., J. S., M.E., L.Y.Y., and E.T.E.; Writing – Original Draft, R.M.S. and S.A.D.; Writing – Review & Editing, R.M.S. and S.A.D.; Supervision, S.A.D.; Funding Acquisition, R.M.S. and S.A.D.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Structural origins of Escherichia coli RNA polymerase open promoter complex stability
Ruth M. Saecker, James Chen, Courtney E. Chiu, Brandon Malone, Johanna Sotiris, Mark Ebrahim, Laura Y. Yen, Edward T. Eng, Seth A. Darst
bioRxiv 2021.09.08.459427; doi: https://doi.org/10.1101/2021.09.08.459427
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Structural origins of Escherichia coli RNA polymerase open promoter complex stability
Ruth M. Saecker, James Chen, Courtney E. Chiu, Brandon Malone, Johanna Sotiris, Mark Ebrahim, Laura Y. Yen, Edward T. Eng, Seth A. Darst
bioRxiv 2021.09.08.459427; doi: https://doi.org/10.1101/2021.09.08.459427

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