| Autor: |
Raushan K. Singh, McKenzie Jonely, Nick A Rejali, Evan Leslie, Rodrigo Noriega, Brenda L. Bass |
| Rok vydání: |
2020 |
| Předmět: |
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| DOI: |
10.1101/2020.09.29.319475 |
| Popis: |
SUMMARYDrosophila melanogasterDicer-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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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