Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region σ 3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

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.