Direct observation of base-pair stepping by RNA polymerase

Nature. 2005 Nov 24;438(7067):460-5. doi: 10.1038/nature04268. Epub 2005 Nov 13.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Pairing*
  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism*
  • DNA-Directed RNA Polymerases / metabolism*
  • Escherichia coli / enzymology*
  • Kinetics
  • Models, Biological
  • Movement*
  • Nucleotides / genetics
  • Nucleotides / metabolism
  • Optics and Photonics
  • Sensitivity and Specificity
  • Templates, Genetic
  • Transcription, Genetic*

Substances

  • Nucleotides
  • DNA
  • DNA-Directed RNA Polymerases