Title | Direct observation of base-pair stepping by RNA polymerase. |
Publication Type | Journal Article |
Year of Publication | 2005 |
Authors | Abbondanzieri, EA, Greenleaf, WJ, Shaevitz, JW, Landick, R, Block, SM |
Journal | Nature |
Volume | 438 |
Issue | 7067 |
Pagination | 460-5 |
Date Published | 2005 Nov 24 |
Keywords | Base Pairing, DNA, DNA-Directed RNA Polymerases, Escherichia coli, Kinetics, Models, Biological, Movement, Nucleotides, Optics and Photonics, Sensitivity and Specificity, Templates, Genetic, Transcription, Genetic |
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 A, 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. |
Alternate Journal | Nature |