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

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. We find that the RPO pathway is branched, generating RPO complexes with different stabilities. 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. 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. ### Competing Interest Statement The authors have declared no competing interest. * 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 SD : standard deviation SE : standard error