Mutagenesis-Based Evidence for an Asymmetric Configuration of the Ring-Shaped Transcription Termination Factor Rho

Abstract : Transcription termination factor Rho is an ATP-dependent ring-shaped molecular motor that tracks along RNA to dissociate RNA DNA duplexes and transcription complexes in its path. The Rho hexamer contains two distinct sites for interaction with RNA. The primary binding site is composed of pyrimidine-specific binding clefts that are located in the N-terminal domains and anchor Rho to transcripts at C-rich Rut (Rho utilization) sites. Components of the secondary binding site (SBS) in the C-terminal domains directly couple RNA binding to ATP hydrolysis in order to translocate RNA through the Rho ring. Published crystal structures of RNA-bound Rho display distinct architectures ('trimer-of-dimers' or asymmetric hexamer) and SBS RNA interaction networks that suggested conflicting models of RNA "handoff" or "escort" by the Rho subunits. To probe the mechanism of mechanochemical transduction in Rho, we have mutated into alanines (or glycines) the residues that make SBS contacts with RNA in the 'trimer-of-dimers' structure supporting the "handoff" model. We find that the resulting single-point mutants have similar RNA binding affinities but exhibit significantly different ATP hydrolysis, transcription termination, and RNA DNA unwinding activities that are more compatible with the asymmetric Rho structure than with the 'trimer-of-dimers' structure and the resulting "handoff" model. We discuss our findings in connection with specific features of the asymmetric Rho structure yet argue that a simple RNA "escort" model is insufficient to account for all experimental evidence. (C) 2010 Elsevier Ltd. All rights reserved.
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Submitted on : Tuesday, July 12, 2011 - 3:27:01 PM
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Makhlouf Rabhi, Veronika Gocheva, Frederique Jacquinot, Andrea Lee, Emmanuel Margeat, et al.. Mutagenesis-Based Evidence for an Asymmetric Configuration of the Ring-Shaped Transcription Termination Factor Rho. Journal of Molecular Biology, Elsevier, 2011, 405 (2), pp.497-518. ⟨10.1016/j.jmb.2010.11.006⟩. ⟨hal-00608241⟩

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