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Direct observation of base-pair stepping by RNA polymerase

Author

Listed:
  • Elio A. Abbondanzieri

    (Department of Applied Physics)

  • William J. Greenleaf

    (Department of Applied Physics)

  • Joshua W. Shaevitz

    (Department of Physics
    University of California)

  • Robert Landick

    (University of Wisconsin)

  • Steven M. Block

    (Department of Applied Physics
    Stanford University)

Abstract

During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with ångström-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7 ± 0.6 Å, a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force–velocity relationship for transcription at both saturating and sub-saturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site.

Suggested Citation

  • Elio A. Abbondanzieri & William J. Greenleaf & Joshua W. Shaevitz & Robert Landick & Steven M. Block, 2005. "Direct observation of base-pair stepping by RNA polymerase," Nature, Nature, vol. 438(7067), pages 460-465, November.
    • Handle: RePEc:nat:nature:v:438:y:2005:i:7067:d:10.1038_nature04268
    DOI: 10.1038/nature04268
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    Cited by:

    1. Antonino Ingargiola & Eitan Lerner & SangYoon Chung & Francesco Panzeri & Angelo Gulinatti & Ivan Rech & Massimo Ghioni & Shimon Weiss & Xavier Michalet, 2017. "Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules," PLOS ONE, Public Library of Science, vol. 12(4), pages 1-27, April.
    2. Lin-Tai Da & Fátima Pardo Avila & Dong Wang & Xuhui Huang, 2013. "A Two-State Model for the Dynamics of the Pyrophosphate Ion Release in Bacterial RNA Polymerase," PLOS Computational Biology, Public Library of Science, vol. 9(4), pages 1-9, April.
    3. Ehsan Akbari & Melika Shahhosseini & Ariel Robbins & Michael G. Poirier & Jonathan W. Song & Carlos E. Castro, 2022. "Low cost and massively parallel force spectroscopy with fluid loading on a chip," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    4. Jordan Douglas & Richard Kingston & Alexei J Drummond, 2020. "Bayesian inference and comparison of stochastic transcription elongation models," PLOS Computational Biology, Public Library of Science, vol. 16(2), pages 1-21, February.
    5. Jan Opfer & Kay-Eberhard Gottschalk, 2012. "Identifying Discrete States of a Biological System Using a Novel Step Detection Algorithm," PLOS ONE, Public Library of Science, vol. 7(11), pages 1-10, November.

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