Transient kinetic studies of the antiviral <i>Drosophila</i> Dicer-2 reveal roles of ATP in self-nonself discrimination.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 33787495.
- Also identified by DOI 10.7554/eLife.65810 and PMC identifier 8079148.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
Some RIG-I-like receptors (RLRs) discriminate viral and cellular dsRNA by their termini, and <i>Drosophila melanogaster</i> 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 RLRs involved in innate immunity.
Medical subject headings
- Adenosine Triphosphate
- Drosophila Proteins
- Drosophila melanogaster
- RNA Helicases
- Ribonuclease III