Some RIG-I-like receptors discriminate viral and cellular dsRNA by their termini, and Drosophila melanogaster Dicer-2 (dmDcr-2) differentially processes dsRNA with blunt or 2 nucleotide 3'-overhanging termini. We investigated the transient kinetic mechanism of the dmDcr-2 reaction using a rapid reaction stopped-flow technique and time-resolved fluorescence spectroscopy. Indeed, we found that ATP binding to dmDcr-2's helicase domain impacts association and dissociation kinetics of dsRNA in a termini-dependent manner, revealing termini-dependent discrimination of dsRNA on a biologically-relevant time-scale (seconds). ATP hydrolysis promotes transient unwinding of dsRNA termini followed by slow rewinding, and directional translocation of the enzyme to the cleavage site. Time-resolved fluorescence anisotropy reveals a nucleotide-dependent modulation in conformational fluctuations (nanoseconds) of the helicase and Platform•PAZ domains that is correlated with termini-dependent dsRNA cleavage. Our study offers a kinetic framework for comparison to other Dicers, as well as all members of the RIG-I- like receptors involved in innate immunity.
- Brenda L Bass
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- James M Berger, Johns Hopkins University School of Medicine, United States
- Received: December 16, 2020
- Accepted: March 31, 2021
- Accepted Manuscript published: March 31, 2021 (version 1)
© 2021, Singh et al.
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