Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes

Yanli Wang, Stefan Juranek, Haitao Li, Gang Sheng, Greg S. Wardle, Thomas Tuschl () and Dinshaw J. Patel ()
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Yanli Wang: Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, New York 10065, USA
Stefan Juranek: Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA
Haitao Li: Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, New York 10065, USA
Gang Sheng: Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, New York 10065, USA
Greg S. Wardle: Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA
Thomas Tuschl: Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA
Dinshaw J. Patel: Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, New York 10065, USA

Nature, 2009, vol. 461, issue 7265, 754-761

Abstract: 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 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 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.

Date: 2009
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DOI: 10.1038/nature08434

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