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.

Keywords: :

• DEAD-box helicase
• CsdA
• DeaD
• StpA
• RNA chaperone
• RNA annealing
• strand displacement
• kinetics
• FRET

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was funded by the University of Vienna and the Austrian Science Fund FWF through the SpecialResearch Program (SFB17) on ‘Modulators of RNA fate and function’, grant F1703 to RS. We thank Bob Zimmermann for proof-reading the manuscript.

Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/rnabiology/article/23475