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Mol Microbiol. 2018 Aug;109(4):445-457. doi: 10.1111/mmi.13983.

Locking the nontemplate DNA to control transcription.

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Department of Microbiology, The Ohio State University, Columbus, OH, 43210.
Center for RNA Biology, The Ohio State University, Columbus, OH, 43210.
Center for RNA Nanobiotechnology and Nanomedicine, The Ohio State University, Columbus, OH, 43210.
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, 43210.
Department of Biochemistry, University of Turku, Turku, FIN-20014, Finland.


Universally conserved NusG/Spt5 factors reduce RNA polymerase pausing and arrest. In a widely accepted model, these proteins bridge the RNA polymerase clamp and lobe domains across the DNA channel, inhibiting the clamp opening to promote pause-free RNA synthesis. However, recent structures of paused transcription elongation complexes show that the clamp does not open and suggest alternative mechanisms of antipausing. Among these mechanisms, direct contacts of NusG/Spt5 proteins with the nontemplate DNA in the transcription bubble have been proposed to prevent unproductive DNA conformations and thus inhibit arrest. We used Escherichia coli RfaH, whose interactions with DNA are best characterized, to test this idea. We report that RfaH stabilizes the upstream edge of the transcription bubble, favoring forward translocation, and protects the upstream duplex DNA from exonuclease cleavage. Modeling suggests that RfaH loops the nontemplate DNA around its surface and restricts the upstream DNA duplex mobility. Strikingly, we show that RfaH-induced DNA protection and antipausing activity can be mimicked by shortening the nontemplate strand in elongation complexes assembled on synthetic scaffolds. We propose that remodeling of the nontemplate DNA controls recruitment of regulatory factors and R-loop formation during transcription elongation across all life.

[Available on 2019-08-01]

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