GSBS Student Publications

Title

Kinetic analysis of the RNAi enzyme complex

GSBS Program

Biochemistry & Molecular Pharmacology

UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology

Date

6-1-2004

Document Type

Article

Medical Subject Headings

Adenosine Triphosphate; Animals; Base Sequence; Catalysis; DNA Primers; Drosophila melanogaster; Kinetics; Protein Binding; *RNA Interference; RNA, Small Interfering; Sequence Homology, Nucleic Acid

Disciplines

Life Sciences | Medicine and Health Sciences

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.

Rights and Permissions

Citation: Nat Struct Mol Biol. 2004 Jul;11(7):599-606. Epub 2004 May 30. Link to article on publisher's site

DOI of Published Version

10.1038/nsmb780

Related Resources

Link to article in PubMed

Journal Title

Nature structural and molecular biology

PubMed ID

15170178