Structural Basis for NusA Stabilized Transcriptional Pausing

Summary

Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, we report single-particle electron cryo-microscopy reconstructions of NusA bound to paused E. coli RNAP elongation complexes with and without a pause-enhancing hairpin in the RNA exit channel. The structures reveal four interactions between NusA and RNAP that suggest how NusA stimulates RNA folding, pausing, and termination. An asymmetric translocation intermediate of RNA and DNA converts the active site of the enzyme into an inactive state, providing a structural explanation for the inhibition of catalysis. Comparing RNAP at different stages of pausing provides insights on the dynamic nature of the process and the role of NusA as a regulatory factor.

Graphical Abstract

Highlights

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  Two cryo-EM reconstructions of paused RNAP elongation complexes bound by NusA

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  NusA provides positively charged cavity for RNA structures and stabilizes pause

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  Asymmetric translocation intermediate explains transcriptional pausing

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  Dynamic process of pausing reflected by RNAP global conformational changes

Abstract

Guo et al. present cryo-EM structures of paused RNAP bound to NusA. NusA interacts through four points with RNAP and forms a positively charged cavity above the pause-stabilizing RNA hairpin. An asymmetric, half-translocated RNA-DNA hybrid (RNA post-translocated, template DNA pre-translocated) explains transcriptional pausing. NusA further stabilizes the paused RNAP.