Kinetic analysis of the RNAi enzyme complex
- 30 May 2004
- journal article
- research article
- Published by Springer Nature in Nature Structural & Molecular Biology
- Vol. 11 (7), 599-606
- https://doi.org/10.1038/nsmb780
Abstract
The siRNA-directed ribonucleoprotein complex, RISC, catalyzes target RNA cleavage in the RNA interference pathway. Here, we show that siRNA-programmed RISC is a classical Michaelis-Menten enzyme in the presence of ATP. In the absence of ATP, the rate of multiple rounds of catalysis is limited by release of the cleaved products from the enzyme. Kinetic analysis suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage, and product release. Bases near the siRNA 5′ end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3′ regions of the siRNA provide a helical geometry required for catalysis. Finally, the position of the scissile phosphate on the target RNA seems to be determined during RISC assembly, before the siRNA encounters its RNA target.Keywords
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