Snapshots of the slicing cycle. The figure shows key structures determined [11-13] and the stages of Ago-mediated silencing they most closely represent. Thermus thermophilus Argonaute (TtAgo) is shown in grey, guide DNA in red and target RNA in blue. The designations given for the structures (for example, 'Apo') are used in the main text and in the other figures. The positions covered by the guide or target strands are listed (numbering from the guide 5' end). Protein data bank (PDB) codes for the structures are as follows: 'Apo' - 3DLB[11], Binary - 3DLH[11], Mismatched ternary - 3F73[12], 12 mer ternary - 3HO1[13], 15 mer ternary - 3HJF[13], 19 mer ternary - 3HK2[13]. The figure, together with Figures 2 and 3, was produced using Pymol [40].

The 'two-state' mechanism and insight into target recognition. (A) Confirmation of the 'two-state' model for guide anchoring. The figure displays the binary complex [11] and early (12 mer ternary) and late (19 mer ternary) propagation complexes [13], illustrating release of the 3' end of the guide (red) from PAZ (green) upon formation of a sufficient number of guide - target base pairs (12-14). Dotted lines indicate connectivity where atoms are invisible in the structures (due to disorder). (B) Exposure of the seed nucleotides in the binary complex. The figure displays the binary complex [11] with the seed nucleotides (red) in a cavity in the narrowed nucleic acid binding channel in TtAgo. The backbone in a quasi-helical conformation is bedded against the MID and PIWI domains (pale green) whilst the base edges face outwards. (C) Regions of seed and 3' supplementary base pairing in the 19 mer ternary complex. The figure highlights base pairing in the 19 mer ternary complex [13] mediated by positions 2-8 of the guide (the seed, in red, with corresponding target nucleotides in blue) and positions 13-16 of the guide (positions of 3' supplementary pairing [26], with guide in pink and target in pale blue).

The slicer catalytic site and a role for the N-domain as a duplex wedge. (A) Assembly of the slicer site. The figure show zoomed-in views of the slicer catalytic site, in the binary [11] and 19 mer ternary [13] complexes. The figure illustrates conformational changes that accompany complementary target strand annealing. Key residues are highlighted. The guide is omitted in the right panel for clarity. The 19 mer ternary complex structure shown was obtained using an N478 catalytic site mutant [13] and, therefore, the structure is partially-distorted with only a single catalytic magnesium ion. (B) The N-domain as a duplex wedge. Two views of the 19 mer ternary complex [13] illustrating encapsulation by TtAgo of the fully annealed complementary guide/target duplex and blockage at position 16 by the N-domain (orange). The view on the left shows the duplex from the guide 5' end and the widened nucleic acid binding channel between the MID/PIWI lobe (pale green) and PAZ (dark green). The view on the right shows the same structure rotated and from above, illustrating enclosure of the duplex and the abrupt arrest at position 16 (guide)/16' (target). Nucleotides 17-21 of the guide and 17'-19' of the target are invisible, presumably disordered, though it is assumed they bypass either side of the N-domain.