Real-Time Single-Molecule Studies of RNA Polymerase–Promoter Open Complex Formation Reveal Substantial Heterogeneity Along the Promoter-Opening Pathway

Anssi M. Malinen*, Jacob Bakermans, Emil Aalto-Setälä, Martin Blessing, David L.V. Bauer, Olena Parilova, Georgiy A. Belogurov, David Dulin, Achillefs N. Kapanidis

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)–σ70 holoenzyme to the promoter DNA. This initial RNAP–promoter closed complex undergoes a series of conformational changes, including the formation of a transcription bubble on the promoter and the loading of template DNA strand into the RNAP active site; these changes lead to the catalytically active open complex (RPO) state. Recent cryo-electron microscopy studies have provided detailed structural insight on the RPO and putative intermediates on its formation pathway. Here, we employ single-molecule fluorescence microscopy to interrogate the conformational dynamics and reaction kinetics during real-time RPO formation on a consensus lac promoter. We find that the promoter opening may proceed rapidly from the closed to open conformation in a single apparent step, or may instead involve a significant intermediate between these states. The formed RPO complexes are also different with respect to their transcription bubble stability. The RNAP cleft loops, and especially the β′ rudder, stabilise the transcription bubble. The RNAP interactions with the promoter upstream sequence (beyond −35) stimulate transcription bubble nucleation and tune the reaction path towards stable forms of the RPO.

Original languageEnglish
Article number167383
Pages (from-to)1-18
Number of pages18
JournalJournal of Molecular Biology
Issue number2
Early online date1 Dec 2021
Publication statusPublished - 30 Jan 2022

Bibliographical note

Funding Information:
This work was supported by Academy of Finland [grant numbers 307775, 314100, 335377 to A.M.M]; Instrumentarium Science Foundation [grant to A.M.M.]; Finnish Cultural Foundation [grants to A.M.M. and O.P.]; the Francis Crick Institute which receives its core funding from Cancer Research UK (FC011104), the UK Medical Research Council (FC011104), and the Wellcome Trust (FC011104) [to D.L.V.B.]; Wellcome Trust grant [grant number 110164/Z/15/Z to A.N.K.]; and UK Biotechnology and Biological Sciences Research Council [grant number BB/H01795X/1 to A.N.K].

Funding Information:
This research was funded in whole, or in part, by the Wellcome Trust [110164/Z/15/Z; FC011104]. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. We thank Richard E. Ebright for providing myxopyronin B. We are grateful to Matti Turtola, Thadée Grocholski and Henri Malmi for constructing plasmids. We thank Abhishek Mazumder for insightful discussions and critical reading of the manuscript. We thank the Cell Imaging and Cytometry core at Turku Bioscience Centre, which is supported by Biocenter Finland, for the access to the microscopes.

Publisher Copyright:
© 2021 The Authors


  • molecular mechanism
  • reaction pathway
  • total internal reflection fluorescence microscopy
  • transcription initiation
  • σ factor


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