Structure of yeast Argonaute with guide RNA

Nature. 2012 Jun 20;486(7403):368-74. doi: 10.1038/nature11211.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Argonaute Proteins / chemistry*
  • Argonaute Proteins / metabolism*
  • Base Sequence
  • Biocatalysis
  • Catalytic Domain
  • Crystallography, X-Ray
  • Eukaryotic Cells / chemistry
  • Eukaryotic Cells / enzymology
  • Fungal Proteins / chemistry*
  • Fungal Proteins / metabolism*
  • Kluyveromyces / chemistry*
  • Kluyveromyces / enzymology
  • Models, Molecular
  • Molecular Conformation
  • Molecular Sequence Data
  • RNA, Guide / chemistry*
  • RNA, Guide / genetics
  • RNA, Guide / metabolism*
  • Saccharomycetales / enzymology
  • Saccharomycetales / genetics

Substances

  • Argonaute Proteins
  • Fungal Proteins
  • RNA, Guide

Associated data

  • GEO/GSE37725
  • PDB/4F1N