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.
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
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).
Pause A, Sonenberg N (1992) Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J 11:2643–2654PubMedCentralPubMedGoogle Scholar
Bizebard T, Ferlenghi I, Iost I et al (2004) Studies on three E. coli DEAD-box helicases point to an unwinding mechanism different from that of model DNA helicases. Biochemistry 43:7857–7866PubMedCrossRefGoogle Scholar
Rasnik I, Mc Kinney SA, Ha T (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3:891–893PubMedCrossRefGoogle Scholar
Hwang H, Kim H, Myong S (2011) Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity. Proc Natl Acad Sci U S A 108:7414–7418PubMedCentralPubMedCrossRefGoogle Scholar
Moreira BG, You Y, Behlke MA et al (2005) Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability. Biochem Biophys Res Commun 327:473–484PubMedCrossRefGoogle Scholar
Putnam A, Jankowsky E (2012) Analysis of duplex unwinding by RNA helicases using stopped-flow fluorescence spectroscopy. Methods Enzymol 511:1–27PubMedCrossRefGoogle Scholar