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Structure of yeast Argonaute with guide RNA

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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 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.

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Figure 1: Cleavage activity of budding-yeast AGO.
Figure 2: KpAGO architecture and copurifying RNA.
Figure 3: Organization of the guide RNA.
Figure 4: An extended, potentially unobstructed nucleic-acid-binding channel in KpAGO.
Figure 5: A plugged-in glutamate finger at the active site.

Accession codes

Primary accessions

Gene Expression Omnibus

Protein Data Bank

Data deposits

The structural coordinates of KpAGO have been deposited in the Protein Data Bank (http://www.rcsb.org/pdb) under accession code 4F1N. RNA-sequencing data have been deposited in the Gene Expression Omnibus (http:// www.ncbi.nlm.nih.gov/geo) under accession number GSE37725.

Change history

  • 22 June 2012

    The HTML published online on 18 May was the non-final version of the proof; this has now been corrected.

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Acknowledgements

We thank K. Rajashankar for data processing and phasing, V. Auyeung and D. Shechner for discussions, the Whitehead Genome Technology Core for high-throughput sequencing, and the NE-CAT beamline at the Advanced Photon Source. This work was supported by National Institutes of Health grants AI068776 (D.J.P.) and GM61835 (D.P.B.), a Human Frontier Science Program Long-term Fellowship (K.N.), a fellowship from the Japan Society for the Promotion of Science for Research Abroad (K.N.), and a National Science Foundation graduate research fellowship (D.E.W.). D.P.B. is an Investigator of the Howard Hughes Medical Institute.

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All authors designed the study and wrote the manuscript. Structural experiments were performed by K.N. under the supervision of D.J.P. Biochemical experiments were performed by D.E.W. under the supervision of D.P.B.

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Correspondence to David P. Bartel or Dinshaw J. Patel.

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This file contains Supplementary Tables 1-6 and Supplementary Figures 1-14. This file has been amended and was replaced on 20 June 2012. (PDF 5212 kb)

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Nakanishi, K., Weinberg, D., Bartel, D. et al. Structure of yeast Argonaute with guide RNA. Nature 486, 368–374 (2012). https://doi.org/10.1038/nature11211

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