Abstact

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 sigma factor sigma(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 sigma3.2 region. This clash triggers a swivel of the RNAP structural module and scrunching of the transcription bubble. We also observe positional rearrangement of the sigma4 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.