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

David Dulin, David L. V. Bauer, Anssi M. Malinen, Jacob J. W. Bakermans, Martin Kaller, Zakia Morichaud, Ivan Petushkov, Martin Depken, Konstantin Brodolin, Andrey Kulbachinskiy, Achillefs N. Kapanidis
doi: https://doi.org/10.1101/199307
David Dulin
1Biological Physics Research Group, Clarendon Laboratory, Department of Physics,University of Oxford, Parks Road, Oxford, OX1 3PU, UK
2Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Hartmannstr. 14, 91052 Erlangen, Germany
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David L. V. Bauer
1Biological Physics Research Group, Clarendon Laboratory, Department of Physics,University of Oxford, Parks Road, Oxford, OX1 3PU, UK
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Anssi M. Malinen
1Biological Physics Research Group, Clarendon Laboratory, Department of Physics,University of Oxford, Parks Road, Oxford, OX1 3PU, UK
3Department of Biochemistry, University of Turku, 20014 Turku, Finland
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Jacob J. W. Bakermans
1Biological Physics Research Group, Clarendon Laboratory, Department of Physics,University of Oxford, Parks Road, Oxford, OX1 3PU, UK
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Martin Kaller
1Biological Physics Research Group, Clarendon Laboratory, Department of Physics,University of Oxford, Parks Road, Oxford, OX1 3PU, UK
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Zakia Morichaud
4Institut de Recherche en Infectiologie de Montpellier (IRIM) UMR9004 CNRS -Université de Montpellier, 1919 Route de Mende, 34293 Montpellier, France
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Ivan Petushkov
5Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia
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Martin Depken
6Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands
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Konstantin Brodolin
4Institut de Recherche en Infectiologie de Montpellier (IRIM) UMR9004 CNRS -Université de Montpellier, 1919 Route de Mende, 34293 Montpellier, France
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Andrey Kulbachinskiy
5Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia
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Achillefs N. Kapanidis
1Biological Physics Research Group, Clarendon Laboratory, Department of Physics,University of Oxford, Parks Road, Oxford, OX1 3PU, UK
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Abstract

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region σ3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

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Posted October 06, 2017.
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Pausing controls branching between productive and non-productive pathways during initial transcription
David Dulin, David L. V. Bauer, Anssi M. Malinen, Jacob J. W. Bakermans, Martin Kaller, Zakia Morichaud, Ivan Petushkov, Martin Depken, Konstantin Brodolin, Andrey Kulbachinskiy, Achillefs N. Kapanidis
bioRxiv 199307; doi: https://doi.org/10.1101/199307
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Pausing controls branching between productive and non-productive pathways during initial transcription
David Dulin, David L. V. Bauer, Anssi M. Malinen, Jacob J. W. Bakermans, Martin Kaller, Zakia Morichaud, Ivan Petushkov, Martin Depken, Konstantin Brodolin, Andrey Kulbachinskiy, Achillefs N. Kapanidis
bioRxiv 199307; doi: https://doi.org/10.1101/199307

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