Wang, Y., Sheng, G., Juranek, S., Tuschl, T. & Patel, D. J. Structure of the guide-strand-containing Argonaute silencing complex. Nature 456, 209-213

Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.
Nature (Impact Factor: 41.46). 09/2008; 456(7219):209-13. DOI: 10.1038/nature07315
Source: PubMed


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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    • "As a general rule, the SS and the 3'-proximal part and 3'-overhang of the AS are more amenable to chemical modification; on the contrary the 5' phosphate, the 5' proximal part and central positions of the AS are more sensitive , especially to multiple or bulky modifications (Bramsen and Kjems, 2013). The 5'- phosphate group of the guide strand is required for the binding to AGO2 (Nykanen et al., 2001; Wang et al., 2008), in addition, the initial interactions between the guide strand and target RNA are mediated by the 5' proximal seed region; therefore modifications of these particular regions may interfere with activity (Wang et al., 2008). Oligonucleotides are synthetized using solid phase phosphoroamidite chemistry. "
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    • "PIWI and other Argonaute proteins can catalyze endonucleolytic cleavage of their targets. Target cleavage requires extensive complementarity between the guide and the target to enable a conformational change that brings the target closer to the Argonaute active site (Wang et al., 2008, 2009). "
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