Structure of yeast Argonaute with guide RNA

The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 Å crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded and processed by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1–8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2′-hydroxyl groups pre-organizing the backbone of nucleotides 2–8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide–target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage. Argonaute proteins are an essential part of the guide-RNA–protein complex that carries out RNA-induced gene silencing; structure–function studies of the yeast complex reveal conserved features of the eukaryotic complex, which underlie formation of the catalytically active conformation. The functional complex that carries out RNA-induced gene silencing consists of an Argonaute (Ago) protein bound to a short single-stranded guide RNA. This complex recognizes and binds a complementary messenger RNA sequence and mediates either RNA cleavage or repression of its translation. The 3.2-Ångström crystal structure of Kluyveromyces polysporus Argonaute, bound by chance to nonspecific guide RNA, has now been determined. The prokaryotic and human Ago protein structures have been solved in inactive conformations, but this yeast Ago structure is in an active conformation that reveals the mechanism of catalysis.