Real-Time Single-Molecule Studies of RNA Polymerase–Promoter Open Complex Formation Reveal Substantial Heterogeneity Along the Promoter-Opening Pathway. Issue 2 (30th January 2022)

• Record Type: Journal Article
• Authors: Malinen, Anssi M.; Bakermans, Jacob; Aalto-Setälä, Emil; Blessing, Martin; Bauer, David L.V.; Parilova, Olena; Belogurov, Georgiy A.; Dulin, David; Kapanidis, Achillefs N.
• Is Part Of: Journal of molecular biology. Volume 434:Issue 2(2022)
• Publication Date: 2022-01-30
• Subjects: transcription initiation -- molecular mechanism -- reaction pathway -- σ factor -- total internal reflection fluorescence microscopy; ALEX Alternating laser excitation -- αCTD C-terminal domain of RNA polymerase α-subunit -- CI confidence interval -- Cor corallopyronin A -- cryo-EM cryo-electron microscopy -- E* apparent FRET efficiency -- FRET fluorescence energy transfer -- GL gate loop in the RNAP β subunit -- HMM Hidden Markov modelling -- LL lid loop in the RNAP β′ subunit -- Myx myxopyronin B -- RL rudder loop in the RNAP β′ subunit -- RNAP RNA polymerase -- RPC RNAP–promoter closed complex -- RPi RNAP–promoter intermediate complex -- RPO RNAP–promoter open complex -- lacCONS consensus lac promoter -- dsLC2 double-stranded consensus lac promoter -- pmLC2 pre-melted consensus lac promoter -- ntDNA non-template DNA -- tDNA template DNA; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2021.167383
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: Highlights: The formation of single RNA polymerase–promoter open complexes probed in real-time. Substantial heterogeneity was found along the promoter-opening pathway. Branched steps include template strand loading into the active site. Branched steps also include transcription bubble stabilisation. RNA polymerase rudder loop stabilises open transcription bubble. Abstract: The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)–σ 70 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 .