Real-Time Observation of Backtracking by Bacterial RNA Polymerase.

ABSTRACT: RNA polymerase (RNAP) backtracking is a backward sliding of the enzyme along DNA and RNA. It plays important roles in many essential processes in bacteria and in eukaryotes. We describe here a fluorescence-based approach that allows a real-time observation of bacterial RNAP backtracking. A Cy3 fluorescence probe, when incorporated into a specific site in the nontemplate strand near the site of backtracking, allows RNAP movements to be monitored near the probe because of a robust enhancement of fluorescence caused by protein proximity. Using this approach, we showed that binding of NTP to the active site prior to phosphodiester bond formation inhibited backtracking, consistent with the coupling of NTP binding to translocation. The extent and the kinetics of backtracking did not show a simpl

SUBMITTER: Lass-Napiorkowska A 

PROVIDER: S-EPMC5921838 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature