Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria
David Dulin (University of Oxford, Friedrich-Alexander-Universität Erlangen-Nürnberg)
David L.V. Bauer (University of Oxford)
Anssi M. Malinen (University of Turku, University of Oxford)
Jacob J.W. Bakermans (University of Oxford)
Martin Kaller (University of Oxford)
Zakia Morichaud (CNRS/Université de Montpellier II)
Ivan Petushkov (Russian Academy of Sciences)
Martin Depken (Kavli institute of nanoscience Delft, TU Delft - Applied Sciences)
Konstantin Brodolin (CNRS/Université de Montpellier II)
Andrey Kulbachinskiy (Russian Academy of Sciences)
Achillefs N. Kapanidis (University of Oxford)
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Abstract
Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription.
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