title: Characterization of the kinetics of RNA annealing and strand displacement activities of the E. coli DEAD-box helicase CsdA.
abstract: CsdA is one of five E. coli DEAD-box helicases and as a cold-shock protein assists RNA structural remodeling at low temperatures. The helicase has been shown to catalyze duplex unwinding in an ATP-dependent way and accelerate annealing of complementary RNAs, but detailed kinetic analyses are missing. Therefore, we performed kinetic measurements using a coupled annealing and strand displacement assay with high temporal resolution to analyze how CsdA balances the two converse activities. We furthermore tested the hypothesis that the unwinding activity of DEAD-box helicases is largely determined by the substrate's thermodynamic stability using full-length CsdA and a set of RNAs with constant length, but increasing GC content. The rate constants for strand displacement did indeed decrease with increasing duplex stability, with a calculated free energy between -31.3 and -40 kcal/mol being the limit for helix unwinding. Thus, our data generally support the above hypothesis, showing that for CsdA substrate thermal stability is an important rate limiting factor.
authors.affiliation: Max F. Perutz Laboratories, Department for Biochemistry, Vienna, Austria.
authors.forename: Sabine
authors.initials: S
authors.lastname: Stampfl
authors.forename: Martina
authors.initials: M
authors.lastname: Doetsch
authors.forename: Mads
authors.initials: M
authors.lastname: Beich-Frandsen
authors.forename: Renée
authors.initials: R
authors.lastname: Schroeder
mesh_terms: D001482:Base Composition; D020029:Base Pairing; D001483:Base Sequence; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007700:Kinetics; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D059370:RNA Folding; D020871:RNA Stability; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; D012330:RNA, Double-Stranded; D012313:RNA; C088840:deaD protein, E coli; D004265:DNA Helicases; D053487:DEAD-box RNA Helicases
keywords: CsdA; DEAD-box helicase; DeaD; FRET; RNA annealing; RNA chaperone; StpA; kinetics; strand displacement