Abstract
RNA polymerase (RNAP) contains a mobile structural module, the ‘clamp,’ that forms one wall of the RNAP active-center cleft and that has been linked to crucial aspects of the transcription cycle, including promoter melting, transcription elongation complex stability, transcription pausing, and transcription termination. Using single-molecule FRET on surface-immobilized RNAP molecules, we show that the clamp in RNAP holoenzyme populates three distinct conformational states and interconvert between these states on the 0.1–1 s time-scale. Similar studies confirm that the RNAP clamp is closed in open complex (RPO) and in initial transcribing complexes (RPITC), including paused initial transcribing complexes, and show that, in these complexes, the clamp does not exhibit dynamic behaviour. We also show that, the stringent-response alarmone ppGpp, which reprograms transcription during amino acid starvation stress, selectively stabilizes the partly-closed-clamp state and prevents clamp opening; these results raise the possibility that ppGpp controls promoter opening by modulating clamp dynamics.
Highlights
RNA polymerase (RNAP) is the main molecular machine responsible for transcription
Single-molecule FRET studies assessing RNAP clamp conformation in solution in freely diffusing single molecules of Escherichia coli RNAP confirmed that the RNAP clamp adopts different conformational states in solution; defined equilibrium population distributions of RNAP clamp states in RNAP core enzyme, RNAP holoenzyme, transcription initiation complexes and transcription elongation complexes; and demonstrated effects of RNAP inhibitors that interact with the RNAP switch region on RNAP clamp conformation [9]
To monitor the conformation of clamp in real-time, we have developed a method of surface-immobilizing doubly labeled RNAP molecules [15,17], and observing the RNAP structure by Single-molecule FRET (smFRET) using a total-internal reflection fluorescence (TIRF) microscope equipped with alternating-laser excitation (ALEX) [15,16,25]
Summary
RNA polymerase (RNAP) is the main molecular machine responsible for transcription. In bacteria, RNAP is a multisubunit protein with an overall shape that resembles a crab claw. Single-molecule FRET (smFRET) studies assessing RNAP clamp conformation in solution in freely diffusing single molecules of Escherichia coli RNAP confirmed that the RNAP clamp adopts different conformational states in solution; defined equilibrium population distributions of RNAP clamp states in RNAP core enzyme, RNAP holoenzyme, transcription initiation complexes and transcription elongation complexes; and demonstrated effects of RNAP inhibitors that interact with the RNAP switch region on RNAP clamp conformation [9] Because those smFRET studies analyzed freely diffusing single molecules––for which it is difficult to monitor individual single molecules over timescales of >10 ms, and it has not been possible to define trajectories of smFRET versus time over timescales of >10 ms––these smFRET studies provided no information about the occurrence, pathway, and kinetics of interconversions between clamp conformational states
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