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RNA polymerase motions during promoter melting

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Science  26 May 2017:
Vol. 356, Issue 6340, pp. 863-866
DOI: 10.1126/science.aam7858

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Trapping RNA polymerase in the act

The enzyme RNA polymerase (RNAP) finds promoter elements in the genome, separates (or “melts”) the DNA strands, and transcribes the template DNA strand to give RNA. A mobile clamp in RNAP plays a key role in initiating transcription. Feklistov et al. locked the clamp of bacterial RNAP in distinct conformations by using small molecules. They then used fluorescent probes to monitor binding as the promoter DNA was separated. Unexpectedly, they found that the clamp transiently closed to nucleate DNA melting, opened to load single-stranded DNA into the active site, and then closed around the template strand to start transcription.

Science, this issue p. 863

Abstract

All cellular RNA polymerases (RNAPs), from those of bacteria to those of man, possess a clamp that can open and close, and it has been assumed that the open RNAP separates promoter DNA strands and then closes to establish a tight grip on the DNA template. Here, we resolve successive motions of the initiating bacterial RNAP by studying real-time signatures of fluorescent reporters placed on RNAP and DNA in the presence of ligands locking the clamp in distinct conformations. We report evidence for an unexpected and obligatory step early in the initiation involving a transient clamp closure as a prerequisite for DNA melting. We also present a 2.6-angstrom crystal structure of a late-initiation intermediate harboring a rotationally unconstrained downstream DNA duplex within the open RNAP active site cleft. Our findings explain how RNAP thermal motions control the promoter search and drive DNA melting in the absence of external energy sources.

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