Abstract
The transcription termination factor Rho from Escherichia coli is a ring-shaped homo-hexameric protein that preferentially interacts with naked cytosine-rich Rut (Rho utilization) regions of nascent RNA transcripts. Once bound to the RNA chain, Rho uses ATP as an energy source to produce mechanical work and disruptive forces that ultimately lead to the dissociation of the ternary transcription complex. Although transcription termination assays have been useful to study Rho activity in various experimental contexts, they do not report directly on Rho mechanisms and kinetics. Here, we describe complementary ATP-dependent RNA–DNA helicase and streptavidin displacement assays that can be used to monitor in vitro Rho’s motor activity in a more direct and quantitative manner.
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Acknowledgments
We apologize to our colleagues, especially past and present members of the Platt, Richardson, Roberts, and von Hippel labs, whose fantastic work on Rho cannot be exhaustively cited here. We gratefully acknowledge J.M. Berger, J.P. Richardson, and P.H. von Hippel for the gift of materials. Special thanks to A.R. Rahmouni, head of the ‘RNP interactions and therapeutic applications’ lab at Centre de Biophysique Moléculaire, for his constant support and stimulating discussions. This work was supported by grants from the Agence Nationale de la Recherche (PCV06-135253), the Conseil Régional du Centre (CPR 07.09.24), the Association pour la Recherche sur le Cancer (grant #3639), and the Ligue contre le Cancer (région Centre).
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Boudvillain, M., Walmacq, C., Schwartz, A., Jacquinot, F. (2009). Simple Enzymatic Assays for the In Vitro Motor Activity of Transcription Termination Factor Rho from Escherichia coli . In: Abdelhaleem, M. (eds) Helicases. Methods in Molecular Biology, vol 587. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-355-8_10
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DOI: https://doi.org/10.1007/978-1-60327-355-8_10
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