Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Argonaute Proteins
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Bacteria / enzymology*
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Binding Sites / genetics
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Catalytic Domain / genetics
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Cations, Divalent / chemistry
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Crystallography, X-Ray
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DNA, Single-Stranded / metabolism
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Endoribonucleases / chemistry*
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Endoribonucleases / genetics
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Endoribonucleases / metabolism
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Eukaryotic Initiation Factor-2
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Eukaryotic Initiation Factors / genetics
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Eukaryotic Initiation Factors / metabolism
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Humans
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Models, Chemical
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Models, Molecular
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Molecular Sequence Data
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Oligonucleotides / metabolism
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Peptide Initiation Factors / genetics
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Peptide Initiation Factors / metabolism
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Protein Binding
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Protein Conformation
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Protein Structure, Tertiary
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RNA, Double-Stranded / metabolism
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RNA, Messenger / metabolism*
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RNA-Induced Silencing Complex / metabolism*
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Sequence Homology, Amino Acid
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Static Electricity
Substances
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AGO1 protein, human
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AGO2 protein, human
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Argonaute Proteins
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Bacterial Proteins
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Cations, Divalent
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DNA, Single-Stranded
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Eukaryotic Initiation Factor-2
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Eukaryotic Initiation Factors
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Oligonucleotides
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Peptide Initiation Factors
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RNA, Double-Stranded
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RNA, Messenger
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RNA-Induced Silencing Complex
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Recombinant Fusion Proteins
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Recombinant Proteins
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Endoribonucleases
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argonaute protein, Aquiflex aeolicus