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.
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Acknowledgements
We thank J. Gelles for general discussions and continued inspiration, D. Bushnell and C. Kaplan for discussions relating to RNAP secondary binding sites, P. Fordyce, N Guydosh, A. Meyer, A. La Porta and M. Woodside for comments on the manuscript, and R. Byer for discussions about the use of helium. W.J.G. acknowledges the support of a Predoctoral Fellowship from the NSF. This work was supported by grants to S.M.B. from the NIH-NIGMS.
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Abbondanzieri, E., Greenleaf, W., Shaevitz, J. et al. Direct observation of base-pair stepping by RNA polymerase. Nature 438, 460–465 (2005). https://doi.org/10.1038/nature04268
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DOI: https://doi.org/10.1038/nature04268
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