Direct observation of base-pair stepping by RNA polymerase

Elio A. Abbondanzieri, William J. Greenleaf, Joshua W. Shaevitz, Robert Landick and Steven M. Block ()
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Elio A. Abbondanzieri: Department of Applied Physics
William J. Greenleaf: Department of Applied Physics
Joshua W. Shaevitz: Department of Physics
Robert Landick: University of Wisconsin
Steven M. Block: Department of Applied Physics

Nature, 2005, vol. 438, issue 7067, 460-465

Abstract: 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.

Date: 2005
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DOI: 10.1038/nature04268

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