title: Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes.
abstract: The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5'-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg(2+)-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide-target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3'-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures.
authors.affiliation: Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, New York 10065, USA.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Wang
authors.forename: Stefan
authors.initials: S
authors.lastname: Juranek
authors.forename: Haitao
authors.initials: H
authors.lastname: Li
authors.forename: Gang
authors.initials: G
authors.lastname: Sheng
authors.forename: Greg S
authors.initials: GS
authors.lastname: Wardle
authors.forename: Thomas
authors.initials: T
authors.lastname: Tuschl
authors.forename: Dinshaw J
authors.initials: DJ
authors.lastname: Patel
mesh_terms: D020029:Base Pairing; D055162:Biocatalysis; D020134:Catalytic Domain / Q000235:genetics; D002413:Cations, Divalent / Q000378:metabolism; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D010766:Phosphorylation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D002413:Cations, Divalent; D034743:RNA-Induced Silencing Complex; D012313:RNA; D004247:DNA; D008274:Magnesium