Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy.

ABSTRACT: During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ⁷⁰-RNAP and the λP_R promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand 'read-out' extends, the RNAP clamp closes, expelling an inhibitory σ⁷⁰ domain from the ac

SUBMITTER: Saecker RM 

PROVIDER: S-EPMC11821292 | biostudies-literature | 2024 Nov

REPOSITORIES: biostudies-literature