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

doi:10.1016/j.jmb.2021.167383

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 Journal › Article › Academic › peer-review

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 (RP_O) state. Recent cryo-electron microscopy studies have provided detailed structural insight on the RP_O 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 RP_O 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 RP_O 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 RP_O.

Original language	English
Article number	167383
Pages (from-to)	1-18
Number of pages	18
Journal	Journal of Molecular Biology
Volume	434
Issue number	2
Early online date	1 Dec 2021
DOIs	https://doi.org/10.1016/j.jmb.2021.167383
Publication status	Published - 30 Jan 2022

Bibliographical note

Publisher Copyright:
© 2021 The Authors

Funding

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, Thad?e 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. All our time-trace data and software used for their analysis will be available from the authors upon reasonable request. 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]. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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]. 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, Thad\u00E9e 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.

Funders	Funder number
Medical Research Council
Instrumentariumin Tiedesäätiö
Suomen Kulttuurirahasto
Turku Bioscience Centre
Biocenter Finland
UK Research and Innovation
Cancer Research UK	FC011104
Wellcome Trust	110164, 110164/Z/15/Z
Biotechnology and Biological Sciences Research Council	BB/H01795X/1
Academy of Finland	335377, 314100, 307775

Keywords

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

Access to Document

10.1016/j.jmb.2021.167383

Persistent URL (handle)

Persistent link

Cite this

Malinen, A. M., Bakermans, J., Aalto-Setälä, E., Blessing, M., Bauer, D. L. V., Parilova, O., Belogurov, G. A., Dulin, D., & 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), 1-18. Article 167383. https://doi.org/10.1016/j.jmb.2021.167383

@article{a6f8678f2f4448c2a1c6551c25cefd41,

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

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.",

keywords = "molecular mechanism, reaction pathway, total internal reflection fluorescence microscopy, transcription initiation, σ factor",

author = "Malinen, \{Anssi M.\} and Jacob Bakermans and Emil Aalto-Set{\"a}l{\"a} and Martin Blessing and Bauer, \{David L.V.\} and Olena Parilova and Belogurov, \{Georgiy A.\} and David Dulin and Kapanidis, \{Achillefs N.\}",

note = "Publisher Copyright: {\textcopyright} 2021 The Authors",

year = "2022",

month = jan,

day = "30",

doi = "10.1016/j.jmb.2021.167383",

language = "English",

volume = "434",

pages = "1--18",

journal = "Journal of Molecular Biology",

issn = "0022-2836",

publisher = "Academic Press",

number = "2",

}

Malinen, AM, Bakermans, J, Aalto-Setälä, E, Blessing, M, Bauer, DLV, Parilova, O, Belogurov, GA, Dulin, D & Kapanidis, AN 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, vol. 434, no. 2, 167383, pp. 1-18. https://doi.org/10.1016/j.jmb.2021.167383

Real-Time Single-Molecule Studies of RNA Polymerase–Promoter Open Complex Formation Reveal Substantial Heterogeneity Along the Promoter-Opening Pathway. / Malinen, Anssi M.; Bakermans, Jacob; Aalto-Setälä, Emil et al.
In: Journal of Molecular Biology, Vol. 434, No. 2, 167383, 30.01.2022, p. 1-18.

Research output: Contribution to Journal › Article › Academic › peer-review

TY - JOUR

T1 - Real-Time Single-Molecule Studies of RNA Polymerase–Promoter Open Complex Formation Reveal Substantial Heterogeneity Along the Promoter-Opening Pathway

AU - Malinen, Anssi M.

AU - Bakermans, Jacob

AU - Aalto-Setälä, Emil

AU - Blessing, Martin

AU - Bauer, David L.V.

AU - Parilova, Olena

AU - Belogurov, Georgiy A.

AU - Dulin, David

AU - Kapanidis, Achillefs N.

PY - 2022/1/30

Y1 - 2022/1/30

N2 - 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.

AB - 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.

KW - molecular mechanism

KW - reaction pathway

KW - total internal reflection fluorescence microscopy

KW - transcription initiation

KW - σ factor

UR - https://www.scopus.com/pages/publications/85121234890

UR - https://www.scopus.com/pages/publications/85121234890#tab=citedBy

U2 - 10.1016/j.jmb.2021.167383

DO - 10.1016/j.jmb.2021.167383

M3 - Article

AN - SCOPUS:85121234890

SN - 0022-2836

VL - 434

SP - 1

EP - 18

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 2

M1 - 167383

ER -

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

Abstract

Bibliographical note

Funding

Keywords

Access to Document

Persistent URL (handle)

Other files and links

Fingerprint

Cite this