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. 2013 Jan 17;288(11):7829–7840. doi: 10.1074/jbc.M112.441030

FIGURE 1.

FIGURE 1.

Binding of guide RNA to Drosophila Argonaute 1 and N-PAZ lobe variants. A, superimposition of T. thermophilus Argonaute structures with imperfect (green, 20-nt target with mismatches at positions 10 and 11; Protein Data Bank code 3F73) or perfect (magenta, 19 base pairs of complementarity; Protein Data Bank code 3DLH) target bound. B, schematic of DmAgo1 PAZ variants. Represented are DmAgo1 variants with N, PAZ, MID, and PIWI domains and linkers (L). DmAgo1-PAZ6 variant contains six point mutations (R352A, K353A, Y354A, T412A, Y413A, and L414A), whereas DmAgo1-ΔN-PAZ lacks the N and PAZ domains. C, competitive double filter binding assays (nitrocellulose and nylon). DmAgo1 was preincubated with radiolabeled 23-nt RNA and unlabeled 23-nt RNA, as homologous inhibitors. Shown is the protein-bound RNA captured on nitrocellulose with different concentrations of competitor. D, apparent Kd values of DmAgo1 variants showing the estimated dissociation constants of the DmAgo1 variants for 23-nt RNA based on competitive filter binding assay. E, competitive binding assay for DmAgo1 variants with radiolabeled 23-nt RNA in the presence of 5-, 15-, or 23-nt RNA competitors.