Advertisement

A FRET-Based, Continuous Assay for the Helicase Activity of DEAD-Box Proteins

  • Thierry BizebardEmail author
  • Marc DreyfusEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

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.

Key words

Helicase assay Fluorescence FRET Continuous enzymatic assay DEAD-box protein Psychrophile RNA 

Notes

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).

References

  1. 1.
    Feller G, Gerday C (2003) Psychrophilic enzymes: hot topics in cold adaptation. Nat Rev Microbiol 1:200–208PubMedCrossRefGoogle Scholar
  2. 2.
    Iost I, Bizebard T, Dreyfus M (2013) Functions of DEAD-box proteins in bacteria: current knowledge and pending questions. Biochim Biophys Acta 1829:866–877PubMedCrossRefGoogle Scholar
  3. 3.
    Cartier G, Lorieux F, Allemand F et al (2010) Cold adaptation in DEAD-box proteins. Biochemistry 49:2636–2646PubMedCrossRefGoogle Scholar
  4. 4.
    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
  5. 5.
    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
  6. 6.
    Rasnik I, Mc Kinney SA, Ha T (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3:891–893PubMedCrossRefGoogle Scholar
  7. 7.
    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
  8. 8.
    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
  9. 9.
    Putnam A, Jankowsky E (2012) Analysis of duplex unwinding by RNA helicases using stopped-flow fluorescence spectroscopy. Methods Enzymol 511:1–27PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Institut de Biologie Physico-chimique, CNRS FRE3630Université Paris DiderotParisFrance

Personalised recommendations