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

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• 作者：Makhlouf Rabhi¹ ; ² ; Veronika Gocheva³ ; ⁴ ; ⁵ ; Fré ; dé ; rique Jacquinot¹ ; Andrea Lee³ ; ⁴ ; ⁵ ; ¹ ; Emmanuel Margeat³ ; ⁴ ; ⁵ ; Marc Boudvillain¹ ; ^{marc.boudvillain@cnrs-orleans.fr}
• 关键词：Rho ; transcription termination ; hexamer ; helicase ; mechanism
• 刊名：Journal of Molecular Biology
• 出版年：2011
• 出版时间：14 January 2011
• 年：2011
• 卷：405
• 期：2
• 页码：497-518
• 全文大小：3382 K

文摘

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.