Bioinformatics and Biochemical Methods to Study the Structural and Functional Elements of DEAD-Box RNA Helicases

  • Josette Banroques
  • N. Kyle TannerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)


DEAD-box RNA helicases have core structures consisting of two, tandemly linked, RecA-like domains that contain all of the conserved motifs involved in binding ATP and RNA, and that are needed for the enzymatic activities. The conserved sequence motifs and structural homology indicate that these proteins share common origins and underlining functionality. Indeed, the purified proteins generally act as ATP-dependent RNA-binding proteins and RNA-dependent ATPases in vitro, but for the most part without the substrate specificity or enzymatic regulation that exists in the cell. We are interested in understanding the relationships between the conserved motifs and structures that confer the commonly shared features, and we are interested in understanding how modifications of the core structure alter the enzymatic properties. We use sequence alignments and structural modeling to reveal regions of interest, which we modify by classical molecular biological techniques (mutations and deletions). We then use various biochemical techniques to characterize the purified proteins and their variants for their ATPase, RNA binding, and RNA unwinding activities to determine the functional roles of the different elements. In this chapter, we describe the methods we use to design our constructs and to determine their enzymatic activities in vitro.

Key words

DEAD-box RNA helicase EMSA ATPase Malachite green Unwinding Structural domains RecA-like Superfamily 2 DExD/H-box 



This work was supported by the Centre National de la Recherche Scientifique, by the HelicaRN [2010 BLAN 1503 01] and HeliDEAD grants [ANR-13-BSV8-0009-01] from the Agence Nationale de la Recherche, by a Programme FPGG032 grant from the Pierre-Gilles de Gennes foundation, and by the Initiative d’Excellence program from the French State [Grant DYNAMO, ANR-11-LABX-0011-01].


  1. 1.
    Byrd AK, Raney KD (2012) Superfamily 2 helicases. Front Biosci (Landmark Ed) 17:2070–2088CrossRefGoogle Scholar
  2. 2.
    Fairman-Williams ME, Guenther UP, Jankowsky E (2010) SF1 and SF2 helicases: family matters. Curr Opin Struct Biol 20:313–324PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Pyle AM (2008) Translocation and unwinding mechanisms of RNA and DNA helicases. Annu Rev Biophys 37:317–336PubMedCrossRefGoogle Scholar
  4. 4.
    Singleton MR, Dillingham MS, Wigley DB (2007) Structure and mechanism of helicases and nucleic acid translocases. Annu Rev Biochem 76:23–50PubMedCrossRefGoogle Scholar
  5. 5.
    Jacob F (1977) Evolution and tinkering. Science 196:1161–1166PubMedCrossRefGoogle Scholar
  6. 6.
    Cordin O, Banroques J, Tanner NK et al (2006) The DEAD-box protein family of RNA helicases. Gene 367:17–37PubMedCrossRefGoogle Scholar
  7. 7.
    Linder P, Jankowsky E (2011) From unwinding to clamping – the DEAD box RNA helicase family. Nat Rev Mol Cell Biol 12:505–516PubMedCrossRefGoogle Scholar
  8. 8.
    Banroques J, Cordin O, Doere M et al (2011) Analyses of the functional regions of DEAD-box RNA “helicases” with deletion and chimera constructs tested in vivo and in vitro. J Mol Biol 413:451–472PubMedCrossRefGoogle Scholar
  9. 9.
    Altschul SF, Madden TL, Schaffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Apweiler R, Martin MJ, O’Donovan C et al (2011) Ongoing and future developments at the Universal Protein Resource. Nucleic Acids Res 39:D214–D219CrossRefGoogle Scholar
  11. 11.
    Rost B, Yachdav G, Liu J (2004) The PredictProtein server. Nucleic Acids Res 32:W321–W326PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Guex N, Peitsch MC, Schwede T (2009) Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis 30:S162–S173PubMedCrossRefGoogle Scholar
  13. 13.
    Hirose S, Shimizu K, Kanai S et al (2007) POODLE-L: a two-level SVM prediction system for reliably predicting long disordered regions. Bioinformatics 23:2046–2053PubMedCrossRefGoogle Scholar
  14. 14.
    Linding R, Russell RB, Neduva V et al (2003) GlobPlot: exploring protein sequences for globularity and disorder. Nucleic Acids Res 31:3701–3708PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Obenauer JC, Cantley LC, Yaffe MB (2003) Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res 31:3635–3641PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Finn RD, Mistry J, Tate J et al (2010) The Pfam protein families database. Nucleic Acids Res 38:D211–D222PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Chan KM, Delfert D, Junger KD (1986) A direct colorimetric assay for Ca2+ -stimulated ATPase activity. Anal Biochem 157:375–380PubMedCrossRefGoogle Scholar
  18. 18.
    Pugh GE, Nicol SM, Fuller-Pace FV (1999) Interaction of the Escherichia coli DEAD box protein DbpA with 23S ribosomal RNA. J Mol Biol 292:771–778PubMedCrossRefGoogle Scholar
  19. 19.
    Panuska JR, Goldthwait DA (1980) A DNA-dependent ATPase from T4-infected Escherichia coli. Purification and properties of a 63,000-dalton enzyme and its conversion to a 22,000-dalton form. J Biol Chem 255:5208–5214PubMedGoogle Scholar
  20. 20.
    Kiianitsa K, Solinger JA, Heyer WD (2003) NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities. Anal Biochem 321:266–271PubMedCrossRefGoogle Scholar
  21. 21.
    Tanner NK, Cordin O, Banroques J et al (2003) The Q motif: a newly identified motif in DEAD box helicases may regulate ATP binding and hydrolysis. Mol Cell 11:127–138PubMedCrossRefGoogle Scholar
  22. 22.
    Turner DH, Mathews DH (2010) NNDB: the nearest neighbor parameter database for predicting stability of nucleic acid secondary structure. Nucleic Acids Res 38:D280–D282PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Sengoku T, Nureki O, Nakamura A et al (2006) Structural basis for RNA unwinding by the DEAD-box protein Drosophila Vasa. Cell 125:287–300PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Institut de Biologie Physico-chimique, CNRS FRE3630ParisFrance
  2. 2.Université Paris DiderotParisFrance

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