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Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex

Nature volume 456, pages 921926 (18 December 2008) | Download Citation

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Abstract

Here we report on a 3.0 Å crystal structure of a ternary complex of wild-type Thermus thermophilus argonaute bound to a 5′-phosphorylated 21-nucleotide guide DNA and a 20-nucleotide target RNA containing cleavage-preventing mismatches at the 10–11 step. The seed segment (positions 2 to 8) adopts an A-helical-like Watson–Crick paired duplex, with both ends of the guide strand anchored in the complex. An arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in an inactive conformation, is released on ternary complex formation. The nucleic-acid-binding channel between the PAZ- and PIWI-containing lobes of argonaute widens on formation of a more open ternary complex. The relationship of structure to function was established by determining cleavage activity of ternary complexes containing position-dependent base mismatch, bulge and 2′-O-methyl modifications. Consistent with the geometry of the ternary complex, bulges residing in the seed segments of the target, but not the guide strand, were better accommodated and their complexes were catalytically active.

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Change history

  • 17 February 2008

    In the version of this article initially published online, the equation in the Methods section was incorrect. The correct equation is shown. The error has been corrected for all versions of the article.

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Protein Data Bank

Data deposits

The structural coordinates of the ternary complex of T. thermophilus Ago bound to 5′-phosphorylated 21-nucleotide guide DNA and 20-nucleotide target RNA have been submitted to the Protein Data Bank under accession number 3F73.

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Acknowledgements

The research was supported by funds from the National Institutes of Health and the Starr Foundation to D.J.P. and T.T. We would like to thank the staff of NE-CAT beam line at the Advanced Photon Source, Argonne National Laboratory, supported by the US Department of Energy, for assistance with data collection.

Author Contributions Y.W. and G.S. expressed and purified T. thermophilus Ago, and grew crystals of the ternary complex. H.L. and Y.W. collected X-ray diffraction data on the micro-focus beam line, and Y.W. solved the structure of the ternary complex. The structural studies were undertaken with the supervision of D.J.P. S.J. was responsible for the cleavage assays on Ago with modified guide strands under the supervision of T.T. D.J.P. and T.T. were primarily responsible for writing the paper and all authors read and approved the submitted manuscript.

Author information

Affiliations

  1. Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Yanli Wang
    • , Haitao Li
    • , Gang Sheng
    •  & Dinshaw J. Patel
  2. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA

    • Stefan Juranek
    •  & Thomas Tuschl

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Corresponding authors

Correspondence to Thomas Tuschl or Dinshaw J. Patel.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Tables 1-3 and Supplementary Figures 1-14 with Legends.

Videos

  1. 1.

    Supplementary Movie 1

    Supplementary Movie 1 shows the Ago ternary complex structure and the conformational transitions on proceeding from the binary to the ternary complex.

  2. 2.

    Supplementary Movie 2

    Supplementary Movie 2 shows the Ago ternary complex structure and the conformational transitions on proceeding from the binary to the ternary complex.

  3. 3.

    Supplementary Movie 3

    Supplementary Movie 3 shows the Ago ternary complex structure and the conformational transitions on proceeding from the binary to the ternary complex.

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DOI

https://doi.org/10.1038/nature07666

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