Abstract
Enzymes from cold-adapted organisms are generally endowed with lower activation enthalpies than their counterparts from organisms growing at higher temperatures, making them better catalysts in the cold. However, the enzymes of RNA metabolism have not been examined in this respect. A challenge for studying cold adaptation of DEAD-box RNA helicases is the low precision of the classical, discontinuous helicase assay based on electrophoretic separation of duplexes and isolated strands. Here, we describe a continuous, FRET-based assay that allows the measurement of the helicase activities of DEAD-box proteins with a precision high enough to detect changes in activation enthalpies associated with cold adaptation.
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Acknowledgments
This work has been supported by the Centre National de la Recherche Scientifique, by the Agence Nationale pour la Recherche [Grants NT05-1_44659 (CARMa), 08-BLAN-0086-02 (mRNases), and 2010 BLAN 1503 01 (HelicaRN) to M.D.], and by the “Investissement d’Avenir” program from the French State (Grant “DYNAMO,” ANR-11-LABX-0011-01).
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Bizebard, T., Dreyfus, M. (2015). A FRET-Based, Continuous Assay for the Helicase Activity of DEAD-Box Proteins. In: Boudvillain, M. (eds) RNA Remodeling Proteins. Methods in Molecular Biology, vol 1259. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2214-7_13
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DOI: https://doi.org/10.1007/978-1-4939-2214-7_13
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