Kinetic Mechanism of Duplex rRNA Unwinding by the DEAD-box Protein, DbpA

Abstract

DEAD-box RNA helicases are enzymes that couple cycles of ATP binding, hydrolysis and product release to the unwinding of duplex RNA. We have previously determined the rate and equilibrium constants defining the ATPase cycle of DbpA, a DEAD-box protein from E. coli that is specifically activated by rRNA. In this study, we have measured rRNA duplex unwinding using a real-time fluorescence assay. Efficient and rapid unwinding of an 8 base pair duplex RNA requires chemical cleavage of ATP. Strand displacement coincides with product release step. Collectively, our analysis allows us to determine how the unwinding kinetic intermediates are coupled to specific ATPase cycle transitions. Because DEAD-box proteins are highly conserved among prokaryotes and eukaryotes, these results will be applicable to eukaryotic DEAD box RNA helicases involved in fundamental aspects of RNA metabolism in the cell.