Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex

Abstract

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. Argonaute or 'Ago' proteins are the 'slicer' components of RISC, the RNA-induced silencing protein complex at the heart of the gene silencing mechanism known as RNA interference (RNAi). The structure of a thermophilic Ago protein bound to a duplex nucleic acid that mimics the interaction of the single-stranded small RNA and the target mRNA has now been determined. The structure reveals that the catalytic cycle is driven by conformational changes in both Argonaute and its bound template. The structure of a thermophilic Ago protein bound to a duplex nucleic acid that mimics the interaction of the single-strand of the small RNA and the target mRNA has been solved. This structure reveals the conformational changes that are necessary to accommodate the target, and the changes that occur in the vicinity of the site of cleavage.