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This content will become publicly available on July 20, 2015

Title: Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

Two components of the RNA polymerase (RNAP) catalytic center, the bridge helix and the trigger loop (TL), have been linked with changes in elongation rate and pausing. Here, single molecule experiments with the WT and two TL-tip mutants of the Escherichia coli enzyme reveal that tip mutations modulate RNAP’s pause-free velocity, identifying TL conformational changes as one of two rate-determining steps in elongation. Consistent with this observation, we find a direct correlation between helix propensity of the modified amino acid and pause-free velocity. Moreover, nucleotide analogs affect transcription rate, suggesting that their binding energy also influences TL folding. A kinetic model in which elongation occurs in two steps, TL folding on nucleoside triphosphate (NTP) binding followed by NTP incorporation/pyrophosphate release, quantitatively accounts for these results. The TL plays no role in pause recovery remaining unfolded during a pause. The model suggests a finely tuned mechanism that balances transcription speed and fidelity.
Authors:
 [1] ;  [2] ;  [3]
  1. Univ. of California, Berkeley, CA (United States)
  2. New York Univ. School of Medicine, New York, NY (United States)
  3. Univ. of California, Berkeley, CA (United States); Kavli Energy Nanosciences Institute at Berkeley, Berkeley, CA (United States)
Publication Date:
OSTI Identifier:
1235103
Grant/Contract Number:
AC03-76SF00098(MSD KC261).; R01-GM107329; R01-GM0325543; AC0376SF00098(MSD KC261).
Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 112; Journal Issue: 3; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Advanced Research Projects Agency - Energy (ARPA-E)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; transcription; single molecule; RNA polymerase; trigger loop; optical tweezers