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Proc Natl Acad Sci U S A. 2015 Jan 20;112(3):743-8. doi: 10.1073/pnas.1421067112. Epub 2014 Dec 31.

Trigger loop folding determines transcription rate of Escherichia coli's RNA polymerase.

Author information

1
Jason L. Choy Laboratory of Single-Molecule Biophysics, the California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720;
2
Department of Biochemistry and Molecular Pharmacology and Howard Hughes Medical Institute, New York University School of Medicine, New York, NY 10016;
3
Jason L. Choy Laboratory of Single-Molecule Biophysics, the California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720; Department of Molecular and Cell Biology, Department of Physics, Department of Chemistry, Biophysics Graduate Group and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; and Kavli Energy Nanosciences Institute at Berkeley, Berkeley, CA 94720 carlos@alice.berkeley.edu.

Abstract

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. This model suggests a finely tuned mechanism that balances transcription speed and fidelity.

KEYWORDS:

RNA polymerase; optical tweezers; single molecule; transcription; trigger loop

PMID:
25552559
PMCID:
PMC4311812
DOI:
10.1073/pnas.1421067112
[Indexed for MEDLINE]
Free PMC Article

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