Abstract In bacteria, RNA polymerase (RNAP) often pauses during the early stages of transcription initiation. The structural basis for these transient pauses remains unclear. Here, we present cryo-electron microscopy (cryo-EM) structures of the paused initiation complex (PIC) and initiation complex (IC) of Mycobacterium tuberculosis ( Mtb ), which include the RNAP core enzyme, the ECF σ factor σ E , transcription factor CarD, promoter DNA, and nascent RNA. Our structures with pre-melted scaffolds reveal an intermediate at the 6–7 nt stage compatible with a paused-like intermediate, associated with steric hindrance between the emerging RNA and the σ3.2 region. This clash triggers a swivel of the RNAP structural module and scrunching of the transcription bubble. We also observe positional rearrangement of the σ4 domain, suggesting a poised pre-escape state. In addition, complementary reconstructions with fully matched DNA scaffolds (N-IC and N-PIC) support the physiological relevance of the captured intermediates. Together, our results support the existence of a mechanistic checkpoint during transcription initiation and suggest an RNA-induced model how RNAP conformational dynamics regulate early transcription.
Zheng et al. (Fri,) studied this question.