The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, whose PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. We report on structures of ternary complexes of T. thermophilus Ago catalytic mutants with 5′-phosphorylated 21-nt guide DNA and complementary target RNAs of length 12-, 15- and 19-nt, which define the molecular basis for Mg2+-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 helix requiring 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 revealed the importance of structural and catalytic divalent metal ions observed in the crystal structures.

Here we report on a 3.0 Å crystal structure of a ternary complex of wild-type Thermus thermophilus argonaute bound to a 5′-phosphorylated 21-nucleotide guide DNA and a 20-nucleotide target RNA containing cleavage-preventing mismatches at the 10-11 step. The seed segment (positions 2 to 8) adopts an A-helical-like Watson-Crick paired duplex, with both ends of the guide strand anchored in the complex. An arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in an inactive conformation, is released on ternary complex formation. The nucleic-acid-binding channel between the PAZ-and PIWI-containing lobes of argonaute widens on formation of a more open ternary complex. The relationship of structure to function was established by determining cleavage activity of ternary complexes containing position-dependent base mismatch, bulge and 2′-O-methyl modifications. Consistent with the geometry of the ternary complex, bulges residing in the seed segments of the target, but not the guide strand, were better accommodated and their complexes were catalytically active.RNA-induced silencing complex (RISC)-associated argonaute (Ago) proteins composed of PAZ-and PIWI-containing modules have a central role in mediating distinct assembly and cleavage steps of the RNA interference (RNAi) catalytic cycle [1][2][3][4] . The Ago protein, as the sole component of RISC exhibiting RNA 'slicer' activity [5][6][7] , is a critical player in the RNAi pathway 8 , effecting transcriptional and post-transcriptional gene regulation in plants and animals [1][2][3][4] . In this capacity, Agos have essential roles ranging from maintaining genomic integrity to heterochromatin formation. Some Ago proteins with active endonuclease domains contribute to the maturation of bound short interfering RNAs (siRNAs) by degradative cleavage of the passenger strand and subsequent guide-strand-mediated sequence-specific ©2008 Macmillan Publishers Limited. All rights reservedCorrespondence and requests for materials should be addressed to D.J.P. (pateld@mskcc.org) or T.T. (ttuschl@mail.rockefeller.edu). Author Contributions Y.W. and G.S. expressed and purified T. thermophilus Ago, and grew crystals of the ternary complex. H.L. and Y.W. collected X-ray diffraction data on the micro-focus beam line, and Y.W. solved the structure of the ternary complex. The structural studies were undertaken with the supervision of D.J.P. S.J. was responsible for the cleavage assays on Ago with modified guide strands under the supervision of T.T. D.J.P. and T.T. were primarily responsible for writing the paper and all authors read and approved the submitted manuscript. Author InformationThe structural coordinates of the ternary complex of T. thermophilus Ago bound to 5′-phosphorylated 21-nucleotide guide DNA and 20-nucleotide target RNA have been submitted to the Protein Data Bank under accession number 3F73. Reprints and permissions information is available at www.nature.com/reprints.Supplementary Information is linked to the online version of...

The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5′-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson–Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas two critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5′-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3′-end-binding pocket made up of the PAZ domain show little effect.

Bacteria acquire memory of viral invaders by incorporating invasive DNA sequence elements into the host CRISPR locus, generating a new spacer within the CRISPR array. We report on the structures of Cas1-Cas2-dual-forked DNA complexes in an effort toward understanding how the protospacer is sampled prior to insertion into the CRISPR locus. Our study reveals a protospacer DNA comprising a 23-bp duplex bracketed by tyrosine residues, together with anchored flanking 3' overhang segments. The PAM-complementary sequence in the 3' overhang is recognized by the Cas1a catalytic subunits in a base-specific manner, and subsequent cleavage at positions 5 nt from the duplex boundary generates a 33-nt DNA intermediate that is incorporated into the CRISPR array via a cut-and-paste mechanism. Upon protospacer binding, Cas1-Cas2 undergoes a significant conformational change, generating a flat surface conducive to proper protospacer recognition. Here, our study provides important structure-based mechanistic insights into PAM-dependent spacer acquisition.