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Direct observation of base-pair stepping by RNA polymerase

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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Figure 1: Experimental set-up, passive force clamp and sensitivity of the RNAP dumbbell assay.
Figure 2: RNAP moves in discrete steps.
Figure 3: Alternative kinetic models for RNAP translocation.
Figure 4: RNAP backstepping and backtracking resolved at high resolution.
Figure 5: Force–velocity and Michaelis–Menten relationships along with model fits.

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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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Correspondence to Steven M. Block.

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This file contains Supplementary Figures 1–5, Supplementary Methods and Supplementary Tables 1–2. (PDF 623 kb)

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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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