Influence of substrate composition on the helicase activity of transcription termination factor Rho: reduced processivity of Rho hexamers during unwinding of RNA-DNA hybrid regions

Abstract : Transcription termination factor Rho forms ring-shaped hexameric structures that load onto segments of the nascent RNA transcript that are C-rich and mostly single-stranded. This interaction converts Rho hexamers into active molecular motors that use the energy resulting from their ATP hydrolase activity to move towards the transcript 3'-end. Upon translocation along the RNA chain, Rho can displace physical roadblocks, such as those formed by RNA-DNA helices, a feature that is likely central to the transcription termination mechanism. To study this "translocase" (helicase) activity, we have designed a collection of Rho substrate chimeras containing an RNA-DNA helix located at various positions with respect to a short (47 nucleotides) artificial loading site. We show that these synthetic constructs represent interesting model substrates able to engage in a productive interaction with Rho and to direct NTP-dependent [5'-->3']-translocation of the hexamers. Using both single and multiple-cycle experimental set-ups, we have also found that Rho helicase activity is strongly dependent on the substrate composition and reaction conditions. For this reason, the rate-limiting step of the helicase reaction could not be identified unambiguously. Yet, the linear dependence of the reaction rate on the hybrid length suggests that helicase action on the RNA-DNA region could be controlled by a unique slow step such as Rho activation, conformational rearrangement, or DNA release. Moreover, removal of the DNA strand occurred at a significant cost for the Rho enzyme, inducing, on average, dissociation from the substrate for every 60-80 base-pairs of hybrid unwound. These results are discussed in relation to the known requirements for Rho substrates, general features of hexameric helicases, and current models for Rho-dependent transcription termination.
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https://hal.archives-ouvertes.fr/hal-00113090
Contributor : Isabelle Frapart <>
Submitted on : Friday, November 10, 2006 - 3:27:55 PM
Last modification on : Monday, May 13, 2019 - 12:34:08 PM

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C. Walmacq, A.R. Rahmouni, M. Boudvillain. Influence of substrate composition on the helicase activity of transcription termination factor Rho: reduced processivity of Rho hexamers during unwinding of RNA-DNA hybrid regions. Journal of Molecular Biology, Elsevier, 2004, 342 (2), pp.403-420. ⟨10.1016/j.jmb.2004.07.026⟩. ⟨hal-00113090⟩

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