Direct observation of base-pair stepping by RNA polymerase.
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.
Abbondanzieri
Elio A
EA
Department of Applied Physics, Stanford University, Stanford, California 94305, USA.
Greenleaf
William J
WJ
Shaevitz
Joshua W
JW
Landick
Robert
R
Block
Steven M
SM
D020029:Base Pairing; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008954:Models, Biological; D009068:Movement; D009711:Nucleotides / Q000235:genetics / Q000378:metabolism; D055095:Optics and Photonics; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic
D009711:Nucleotides; D004247:DNA; D012321:DNA-Directed RNA Polymerases