Structural basis of pausing during transcription initiation in mycobacterium tuberculosis

TL;DR

This study reveals how RNA polymerase pauses during early transcription in Mycobacterium tuberculosis, using cryo-EM structures to show interactions that create a checkpoint.

Contribution

The study provides the first structural insights into the paused initiation complex in Mtb, revealing RNA-induced conformational dynamics.

Findings

Cryo-EM structures show a paused intermediate at the 6–7 nt stage with steric hindrance between RNA and σ3.2.

Structural rearrangements suggest a poised pre-escape state involving the σ4 domain.

Complementary reconstructions confirm the physiological relevance of the captured intermediates.

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 σE, 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.

Pausing during early transcription by bacterial RNA polymerase is not fully understood. Here, the authors determine the structures of the paused initiation complex in Mycobacterium tuberculosis, revealing how RNA and polymerase interactions create a transcription checkpoint.

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