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Nat Struct Mol Biol. 2004 Jul;11(7):599-606. Epub 2004 May 30.

Kinetic analysis of the RNAi enzyme complex.

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1
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

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.

PMID:
15170178
DOI:
10.1038/nsmb780
[Indexed for MEDLINE]
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