Purified Argonaute2 and an siRNA form recombinant human RISC

Rivas, Fabiola V., Tolia, Nirja H., Song, Ji-Joon, Aragon, Juan P., Liu, Jidong, Hannon, Gregory J., Joshua-Tor, Leemor (2005) Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct Mol Biol, 12 (4). pp. 340-9. ISSN 1545-9993 (Print) (Public Dataset)

URL: http://www.ncbi.nlm.nih.gov/pubmed/15800637
DOI: 10.1038/nsmb918

Abstract

Genetic, biochemical and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector complex, RISC. Here we show that recombinant, human Argonaute2 can combine with a small interfering RNA (siRNA) to form minimal RISC that accurately cleaves substrate RNAs. Recombinant RISC shows many of the properties of RISC purified from human or Drosophila melanogaster cells but also has surprising features. It shows no stimulation by ATP, suggesting that factors promoting product release are missing from the recombinant enzyme. The active site is made up of a unique Asp-Asp-His (DDH) motif. In the RISC reconstitution system, the siRNA 5' phosphate is important for the stability and the fidelity of the complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex.

Item Type: Paper
Uncontrolled Keywords: Adenosine Triphosphate metabolism pharmacology Amino Acid Motifs Animals Base Sequence Binding Sites Cell Line Crystallography X-Ray Drosophila melanogaster Humans Kinetics Models Molecular Molecular Sequence Data Peptide Initiation Factors chemistry genetics isolation purification metabolism Phosphates metabolism Protein Folding Protein Structure Tertiary RNA Small Interfering chemistry genetics metabolism RNA-Induced Silencing Complex chemistry genetics metabolism Recombinant Proteins metabolism Ribonuclease H Calf Thymus chemistry Structural Homology Protein
Subjects: Investigative techniques and equipment > X-Ray Diffraction
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > argonaute proteins
CSHL Authors:
Communities: CSHL labs > Hannon lab
CSHL labs > Joshua-Tor lab
Watson School > Publications
Depositing User: CSHL Librarian
Date Deposited: 06 Jan 2012 16:03
Last Modified: 05 Sep 2017 18:33
Related URLs:
Dataset ID:
URI: http://repository.cshl.edu/id/eprint/22686

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving