The formation of the bacterial RNA polymerase-promoter open complex involves a branched pathway

Malinen, A. M.; Bakermans, J.; Aalto-Setälä, E.; Blessing, M.; Bauer, D. L.; Parilova, O.; Belogurov, G. A.; Dulin, D.; Kapanidis, A. N.

doi:10.1101/2021.03.27.437306

bioRxiv · 10.1101/2021.03.27.437306

The formation of the bacterial RNA polymerase-promoter open complex involves a branched pathway

Malinen, A. M.·Bakermans, J.·Aalto-Setälä, E.·Blessing, M.·Bauer, D. L.·Parilova, O.·Belogurov, G. A.·Dulin, D.·Kapanidis, A. N.

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Abstract

The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)-{sigma}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. We find that the RPO pathway is branched, generating RPO complexes with different stabilities. The RNAP cleft loops, and especially the {beta} 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. The mechanistic heterogeneity of the RPO pathway may be a prerequisite for its regulation since such heterogeneity allows the amplification of small promoter sequence or transcription-factor-dependent changes in the free energy profile of the RPO pathway to large differences in transcription efficiency.

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Malinen, A. M., Bakermans, J., Aalto-Setälä, E., Blessing, M., Bauer, D. L., Parilova, O., Belogurov, G. A., Dulin, D., Kapanidis, A. N.. 2021-03-28. The formation of the bacterial RNA polymerase-promoter open complex involves a branched pathway. https://doi.org/10.1101/2021.03.27.437306

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