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A stepwise 2′-hydroxyl activation mechanism for the bacterial transcription termination factor Rho helicase

Nature Structural & Molecular Biology volume 16, pages 1309–1316 (2009)Cite this article

Abstract

The bacterial Rho factor is a ring-shaped ATP-dependent helicase that tracks along RNA transcripts and disrupts RNA-DNA duplexes and transcription complexes in its path. Using combinatorial nucleotide analog interference mapping (NAIM), we explore the topology and dynamics of functional Rho–RNA complexes and reveal the RNA-dependent stepping mechanism of Rho helicase. Periodic Gaussian distributions of NAIM signals show that Rho forms uneven productive interactions with the track nucleotides and disrupts RNA-DNA duplexes in a succession of large (∼7-nucleotide-long) discrete steps triggered by 2′-hydroxyl activation events. This periodic 2′-OH–dependent activation does not depend on the RNA-DNA pairing energy but is finely tuned by sequence-dependent interactions with the RNA track. These features explain the strict RNA specificity and contextual efficiency of the enzyme and provide a new paradigm for conditional tracking by a helicase ring.

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Figure 1: NAIM of Rho-RNA interactions.
Figure 2: Summary of interference effects in RanchorRtrack34D34.
Figure 3: Extended periodicity of 2′-deoxy effects in the tracking strand of a longer RNA-DNA substrate (HybA).
Figure 4: Stepwise 2′-OH–dependent control of forward Rho motion.
Figure 5: Effect of single point SBS mutations on Rho-directed unwinding of HybA.
Figure 6: Possible models accounting for a standard ∼7-nt-long step size between 2′-OH–dependent activation events.

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Acknowledgements

We gratefully acknowledge J.M. Berger (Univ. California, Berkeley), J. Richardson (Indiana Univ.) and P.H. von Hippel (Univ. Oregon) for the gifts of plasmids and batches of purified wild-type Rho, F. Coste for help with molecular graphics, and T. Bizebard, E. Delagoutte, M. Nollmann and C. Royer for critical reading of the manuscript and helpful suggestions. This work was supported by grants from the Agence Nationale de la Recherche (PCV 2006) and the Conseil Régional du Centre (AO 2007).

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Author notes

  1. Institut National de la Santé et de la Recherche Médicale U554, Montpellier, France.

Authors and Affiliations

  1. Centre Nationale de la Recherche Scientifique (CNRS) UPR4301, Centre de Biophysique Moléculaire, Orléans, France

    Annie Schwartz, Makhlouf Rabhi, Frédérique Jacquinot, A Rachid Rahmouni & Marc Boudvillain

  2. Ecole Doctorale Sciences et Technologies, Université d'Orléans, Orléans, France

    Makhlouf Rabhi

  3. CNRS UMR5048, Centre de Biochimie Structurale, Montpellier, France

    Emmanuel Margeat

  4. Universités Montpellier 1 et 2, Montpellier, France

    Emmanuel Margeat

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  1. Annie Schwartz
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Contributions

A.S. performed NAIM experiments and analyzed data; M.R. performed helicase kinetics and analyzed data; F.J. prepared WT and mutant Rho enzymes; E.M. performed QSAR and autocorrelation experiments, analyzed data and wrote the paper; A.R.R. analyzed data and wrote the paper; M.B. performed exploratory experiments, analyzed data and wrote the paper.

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Correspondence to Marc Boudvillain.

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Schwartz, A., Rabhi, M., Jacquinot, F. et al. A stepwise 2′-hydroxyl activation mechanism for the bacterial transcription termination factor Rho helicase. Nat Struct Mol Biol 16, 1309–1316 (2009). https://doi.org/10.1038/nsmb.1711

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