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 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.
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.
Kotaro
K
Nakanishi
David E
DE
Weinberg
David P
DP
Bartel
Dinshaw J
DJ
Patel
D060565:Argonaute Proteins / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005057:Eukaryotic Cells / Q000737:chemistry / Q000201:enzymology; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D007716:Kluyveromyces / Q000737:chemistry* / Q000201:enzymology; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D004718:Saccharomycetales / Q000201:enzymology / Q000235:genetics; D058727:RNA, Small Untranslated
D060565:Argonaute Proteins; D005656:Fungal Proteins; D058727:RNA, Small Untranslated