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Kinetic analysis of the RNAi enzyme complex

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.

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Figure 1: Product release limits the rate of catalysis by RISC.
Figure 2: In the absence of ATP, mismatches between the 3′ end of the siRNA guide strand and the target RNA facilitate product release, but reduce the rate of target cleavage.
Figure 3: Tolerance of RISC for 3′ mismatches.
Figure 4: Limited tolerance of RISC for 5′ mismatches.
Figure 5: Michaelis-Menten and Ki analysis for matched and mismatched siRNAs reveal distinct contributions to binding and catalysis for the 5′, central and 3′ regions of the siRNA.
Figure 6

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Acknowledgements

We thank J. McLachlan for maintaining our fly colony, and members of the Zamore lab, S. Blacklow, T. Carruthers and D. Turner for encouragement, helpful discussions and comments on the manuscript. P.D.Z. is a Pew scholar in the biomedical sciences and a W.M. Keck Foundation young scholar in medical research. This work was supported in part by grants from the US National Institutes of Health to P.D.Z. (GM62862-01 and GM65236-01).

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Correspondence to Phillip D Zamore.

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P.D.Z. is a cofounder of Alnylam Pharmaceuticals, a biotechnology company devoted to the development of RNAi-based therapeutics.

Supplementary information

Supplementary Figure 1

Exogenously programmed RISC is an enzyme. (PDF 46 kb)

Supplementary Figure 2

Michaelis-Menten and competitor analysis of RISC. (PDF 29 kb)

Supplementary Figure 3

RNAs used in this study. (PDF 424 kb)

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Haley, B., Zamore, P. Kinetic analysis of the RNAi enzyme complex. Nat Struct Mol Biol 11, 599–606 (2004). https://doi.org/10.1038/nsmb780

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