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Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage.

Molecular cell | Aug 5, 2005

http://www.ncbi.nlm.nih.gov/pubmed/16061186

Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in "slicer" activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.

Pubmed ID: 16061186 RIS Download

Mesh terms: Amino Acid Sequence | Argonaute Proteins | Bacteria | Bacterial Proteins | Binding Sites | Catalytic Domain | Cations, Divalent | Crystallography, X-Ray | DNA, Single-Stranded | Endoribonucleases | Eukaryotic Initiation Factor-2 | Eukaryotic Initiation Factors | Humans | Models, Chemical | Models, Molecular | Molecular Sequence Data | Oligonucleotides | Peptide Initiation Factors | Protein Binding | Protein Conformation | Protein Structure, Tertiary | RNA, Double-Stranded | RNA, Messenger | RNA-Induced Silencing Complex | Recombinant Fusion Proteins | Recombinant Proteins | Sequence Homology, Amino Acid | Static Electricity

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Associated grants

  • Agency: NCI NIH HHS, Id: P30 CA008748
  • Agency: NIAID NIH HHS, Id: R01 AI068776
  • Agency: NIGMS NIH HHS, Id: R01 GM068476-01

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