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bioRxiv · 10.1101/2023.08.30.555460

Sm-like protein Rof inhibits transcription termination factor Rho by binding site obstruction and conformational insulation

Said, N.·Finazzo, M.·Hilal, T.·Wang, B.·Selinger, T. L.·Gjorgjevikj, D.·Artsimovitch, I.·Wahl, M. C.
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Abstract

Transcription termination factor {rho} is a hexameric, RNA-dependent NTPase that can adopt active closed-ring and inactive open-ring conformations. The Sm-like protein Rof, a homolog of the RNA chaperone Hfq, inhibits {rho}-dependent termination in vivo but recapitulation of this activity in vitro has proven difficult and the precise mode of Rof action is presently unknown. Our electron microscopic structures of {rho}-Rof and {rho}-RNA complexes show that Rof undergoes pronounced conformational changes to bind {rho} at the protomer interfaces, undercutting {rho} conformational dynamics associated with ring closure and occluding extended primary RNA-binding sites that are also part of interfaces between {rho} and RNA polymerase. Consistently, Rof impedes {rho} ring closure, {rho}-RNA interactions, and {rho} association with transcription elongation complexes. Structure-guided mutagenesis coupled with functional assays confirmed that the observed {rho}-Rof interface is required for Rof-mediated inhibition of cell growth and {rho}-termination in vitro. Bioinformatic analyses revealed that Rof is restricted to Pseudomonadota and that the {rho}-Rof interface is conserved. Genomic contexts of rof differ between Enterobacteriaceae and Vibrionaceae, suggesting distinct modes of Rof regulation. We hypothesize that Rof and other cellular anti-terminators silence {rho} under diverse, but yet to be identified, stress conditions when unrestrained transcription termination by {rho} would be lethal.

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Said, N., Finazzo, M., Hilal, T., Wang, B., Selinger, T. L., Gjorgjevikj, D., Artsimovitch, I., Wahl, M. C.. 2023-08-31. Sm-like protein Rof inhibits transcription termination factor Rho by binding site obstruction and conformational insulation. https://doi.org/10.1101/2023.08.30.555460

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