Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria

D. Dulin, D.L.V. Bauer, A.M. Malinen, J.J.W. Bakermans, M. Kaller, Z. Morichaud, I. Petushkov, M. Depken, K. Brodolin, A. Kulbachinskiy, A.N. Kapanidis

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

© 2018 The Author(s).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.
Original languageEnglish
Article number1478
JournalNature Communications
Volume9
Issue number1
DOIs
Publication statusPublished - 1 Dec 2018
Externally publishedYes

Funding

We thank Dr. Francesco Pedaci, Dr. Mohamed Fareh, and Tao Ju Cui for carefully reading the manuscript. We also thank Dr. Jan Lipfert for discussion and Dr. Jun Fan for experimental support during the revision process. This work was supported by grants to ANK from the European Research Council (261227), the Wellcome Trust (110164/Z/15/ Z), and the UK BBSRC (BB/H01795X/1 and BB/J00054X/1). D.L.V.B. was supported by an US National Science Foundation grant (1309306) and by an EPA Cephalosporin Junior Research Fellowship at Linacre College, Oxford. D.D. was supported by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg. A.M.M. was supported by the Instrumentarium Science Foundation, Finnish Cultural Foundation and Alfred Kordelin Foundation. I.P. and A.K. were supported by the Russian Foundation for Basic Research grant 17-54-150009.

FundersFunder number
University of Erlangen-Nuremberg
National Science Foundation1309306
U.S. Environmental Protection Agency
Alfred Kordelinin Säätiö
Wellcome Trust110164/Z/15/ Z
Linacre College, University of Oxford
Biotechnology and Biological Sciences Research CouncilBB/J00054X/1, BB/H01795X/1
European Research Council261227
Russian Foundation for Basic Research17-54-150009
Suomen Kulttuurirahasto
Instrumentariumin Tiedesäätiö
Interdisziplinäres Zentrum für Klinische Forschung, Universitätsklinikum Würzburg

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