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

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

Abstract

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 

Notes

Acknowledgments

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].

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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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