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 angstrom-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. In a paper building on over a decade of progress in single molecule biophysics, Abbondanzieri et al. report the first experiments capable of resolving base-pair steps for an individual enzyme moving along DNA. The enzyme is RNA polymerase, captured by an all-optical force clamp in the act of transcribing RNA from a DNA template. The ability to detect Angstrom-scale motions in a single enzyme opens up new areas of study of biomolecules including perhaps single-molecule DNA sequencing.