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.
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 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].
Linder P, Jankowsky E (2011) From unwinding to clamping – the DEAD box RNA helicase family. Nat Rev Mol Cell Biol 12:505–516PubMedCrossRefGoogle Scholar
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
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
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
Chan KM, Delfert D, Junger KD (1986) A direct colorimetric assay for Ca2+ -stimulated ATPase activity. Anal Biochem 157:375–380PubMedCrossRefGoogle Scholar
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
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
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
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
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
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