A real-time, transient kinetic study of Drosophila melanogaster Dicer-2 elucidates mechanism of termini-dependent cleavage of dsRNA.

Abstract

Drosophila melanogaster Dicer-2 (dmDcr-2) differentially processes dsRNA with blunt or 2 nucleotide 3-overhanging termini. We investigated the transient kinetic mechanism of these reactions using a rapid reaction stopped-flow technique and time-resolved fluorescence spectroscopy. We found that ATP binding to dmDcr-2s helicase domain impacts the kinetics of dsRNA binding and dissociation in a termini-dependent manner, emphasizing the termini-dependent discrimination of dsRNA on a biologically-relevant time-scale. ATP-hydrolysis mediates local unwinding of dsRNA, and directional translocation on unwound single-stranded RNA, which is concurrent with a slow rewinding prior to dsRNA cleavage. Time-resolved fluorescence anisotropy reveals a nucleotide-dependent change in conformational dynamics of the helicase and Platform*PAZ domains in the nanosecond timescale that is correlated with termini-dependent dsRNA cleavage. Our study delineates kinetic events and transient intermediates for a Dicer-catalyzed reaction, thus establishing a framework for understanding other Dicers and how accessory factors modulate the reaction.