Structure of yeast Argonaute with guide RNA

• Main Authors: Nakanishi, Kotaro (Author), Weinberg, David E. (Contributor), Bartel, David (Contributor), Patel, Dinshaw J. (Author)
• Other Authors: Massachusetts Institute of Technology. Department of Biology (Contributor), Whitehead Institute for Biomedical Research (Contributor)
• Format: Article
• Language: English
• Published: Nature Publishing Group, 2013-12-19T19:43:50Z.
• Subjects: Article
• Online Access: Get fulltext

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.
National Institutes of Health (U.S.) (grant AI068776)
National Institutes of Health (U.S.) (grant GM61835)
Human Frontier Science Program (Strasbourg, France) (Long-term Fellowship)
Japan Society for the Promotion of Science
Howard Hughes Medical Institute (Investigator)
National Science Foundation (U.S.) (Graduate Research Fellowship)