Direct observation of base-pair stepping by RNA polymerase

Elio A. Abbondanzieri, William J. Greenleaf, Joshua W. Shaevitz, Robert Landick, Steven M. Block

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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.

Original languageEnglish (US)
Pages (from-to)460-465
Number of pages6
JournalNature
Volume438
Issue number7067
DOIs
StatePublished - Nov 24 2005
Externally publishedYes

All Science Journal Classification (ASJC) codes

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    Abbondanzieri, E. A., Greenleaf, W. J., Shaevitz, J. W., Landick, R., & Block, S. M. (2005). Direct observation of base-pair stepping by RNA polymerase. Nature, 438(7067), 460-465. https://doi.org/10.1038/nature04268