SUMMARY
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-2’s 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.
Competing Interest Statement
The authors have declared no competing interest.
