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

Malinen AM, Bakermans J, Aalto-Setälä E, Blessing M, Bauer DL, Parilova O, Belogurov GA, Dulin D, Kapanidis AN (2022)

Publication Type: Journal article

Publication year: 2022

Journal

Journal of Molecular Biology Elsevier

Book Volume: 434

Article Number: 167383

Journal Issue: 2

DOI: 10.1016/j.jmb.2021.167383

Abstract

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.

Involved external institutions

Turku University Hospital / Turun yliopistollinen keskussairaala (TYKS) FI Finland (FI) University of Oxford GB United Kingdom (GB)

How to cite

APA:

Malinen, A.M., Bakermans, J., Aalto-Setälä, E., Blessing, M., Bauer, D.L., Parilova, O.,... Kapanidis, A.N. (2022). Real-Time Single-Molecule Studies of RNA Polymerase–Promoter Open Complex Formation Reveal Substantial Heterogeneity Along the Promoter-Opening Pathway. Journal of Molecular Biology, 434(2). https://dx.doi.org/10.1016/j.jmb.2021.167383

MLA:

Malinen, Anssi M., et al. "Real-Time Single-Molecule Studies of RNA Polymerase–Promoter Open Complex Formation Reveal Substantial Heterogeneity Along the Promoter-Opening Pathway." Journal of Molecular Biology 434.2 (2022).

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