|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|
|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|
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.