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Structure of the guide-strand-containing argonaute silencing complex

Abstract

The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5′-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson–Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas two critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5′-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3′-end-binding pocket made up of the PAZ domain show little effect.

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Figure 1: Crystal structure of T. thermophilus argonaute bound to a 5′-phosphorylated 21-base DNA guide strand.
Figure 2: Intermolecular hydrogen-bonding alignments in the T. thermophilus Ago bound to a 21-base DNA guide strand.
Figure 3: Conformational changes in T. thermophilus Ago on formation of the 21-base DNA guide strand complex.
Figure 4: DNA-guide-dependent RNA cleavage activity of wild-type and mutant T. thermophilus Ago.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structures of T. thermophilus Ago bound to 5′-phosphorylated 21-base and 10-base DNAs have been deposited to the Protein Data Bank under accession codes 3DLH and 3DLB, respectively.

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Acknowledgements

The research was supported by funds from the NIH to D.J.P. and T.T. We 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 its mutants, and grew crystals of the complex. Y.W. collected X-ray diffraction data and solved the structure of the complex. The structural studies were undertaken under the supervision of D.J.P. S.J. was responsible for the cleavage assays on the wild-type and mutant Agos under the supervision of T.T. All authors read and approved the submitted manuscript.

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Correspondence to Dinshaw J. Patel.

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The file contains Supplementary Text, Supplementary Table 1 and Supplementary Figures 1-12 with Legends. (PDF 8508 kb)

Supplementary Movie 1

The file contains Supplementary Movie 1, rotating Fig. 1c. (AVI 22223 kb)

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The file contains Supplementary Movie 2, which visualizes the transition between Fig. 3a and Fig. 1c. (AVI 4515 kb)

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Wang, Y., Sheng, G., Juranek, S. et al. Structure of the guide-strand-containing argonaute silencing complex. Nature 456, 209–213 (2008). https://doi.org/10.1038/nature07315

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