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. 2017 Jun 27;45(15):9149–9163. doi: 10.1093/nar/gkx547

Figure 1.

Figure 1.

Crystal Structure of TtAgo Bound to 5′-phosphorylated 22-nt Guide DNA and 19-nt Target DNA Containing a 7T8 (TtAgo D546N Catalytic Mutant) and 4A5 (TtAgo Wild-type) Bulges Positioned Within the Seed Segment on the Guide Strand. (A) Sequence of the guide DNA–target DNA duplex (top panel), with the actual alignment of the bulge in the crystal structure of the ternary complex (bottom boxed panel), where a thymine stacks into the duplex between positions 7 and 8 of the guide strand. The traceable segments of the nucleotides of the guide DNA and target DNA in the structure of the ternary complex are shown in red and blue, respectively. The dashed lines show weakened Watson–Crick pairs. (B) 2.9 Å structure of TtAgo (N546 catalytic mutant) bound to 5′-phosphorylated 22-nt guide DNA (in red) and 19-nt target DNA (in blue) containing a 7T8 bulge positioned on the guide strand within the seed segment. There is one molecule of the complex in the asymmetric unit. The PAZ domain is disordered and the 3′-end of the guide strand cannot be monitored in the complex. (C) A stick representation of the bulge site and two flanking base pairs, with the stacked-in thymine highlighted in biscuit color in the 7T8 bulge-containing ternary complex. (D) The positioning of the DNA target strand of the control containing no bulge (in silver) and in the 7T8 bulge (in blue) relative to the catalytic residues (D478, D660, E512 and D546N mutant) of the RNase H fold of the PIWI domain in the TtAgo ternary complex. The catalytic residues are equidistant from the phosphate linking the 10’–11’ step (colored in magenta) in the control (in silver) and 7T8 bulge (in blue)-containing Ago ternary complexes. (E) Superposition of the guide-target duplex containing no bulge (in silver) and 7T8 bulge (in blue) in Ago ternary complexes. The guide strand is labeled g and the 10’–11’ phosphate at the cleavage site on the target strand is indicated by a red arrow. (F) Sequence of the guide DNA–target DNA duplex (top panel), with the actual alignment of the bulge in the crystal structure of the ternary complex (bottom boxed panel), where a adenine stacks into the duplex between positions 4 and 5 of the guide strand. Wild-type TtAgo was used to generate crystals of this complex. (G) A stick representation of the bulge site and two flanking base pairs, with the stacked- in adenine highlighted in biscuit color in the 3.1 Å structure of the 4A5 bulge-containing ternary complex. There is one molecule of the complex in the asymmetric unit and the 3′-end of the guide strand is inserted into the PAZ pocket of an adjacent molecule in the crystal lattice (not shown). (H) The positioning of the DNA target strand of the 4A5 bulge (in blue) relative to the catalytic residues (D478, D660, E512 and D546) of the RNase H fold of the PIWI domain in the wild-type TtAgo ternary complex. Note that the backbone has cleaved at the 10’–11’ step in the target strand and that a pair of Mg2+ cations were identified at the cleavage site in the wild-type TtAgo ternary complex.