Figure 2.

Binding of Argonaute proteins to m⁷GTP-Sepharose reveal allosteric behavior. a, Binding of purified Argonaute MID-domains and PurR protein to the m⁷GTP-Sepharose (Dm, D. melanogaster; Ce, C. elegans; Hs, H. sapiens; Ec, E. coli). miRNA and siRNA related Argonautes are shown in separate boxes. MBP is used as a negative control. b, Binding of DmAgo1-MID and DmAgo2-MID domains to m⁷GTP-Sepharose in the presence of GTP. The total amount of nucleotide is 500 μM, input (10%) and bound fraction (40%) were analyzed by western blotting. c, Binding of DmAgo1 and CeAlg1 MID domains to m⁷GTP-Sepharose is stimulated by addition of 250 μM GTP. Input (10%) and bound fraction (50%) were analyzed by western blotting. d, Binding of full-length DmAgo1, and not of DmAgo2ΔQ, to m⁷GTP-Sepharose is specific and stimulated by addition of short RNA (23-mer). Binding to m⁷GTP-resin is competed by addition of m⁷GpppG. Binding of both, DmAgo1 and DmAgo2ΔQ, to GTP- and GMP-Sepharose is competed by addition of short RNA (23-mer). m⁷GpppG shows no effect on binding to these resins. Input (20%) and bound fraction (25%) were analyzed by western blotting. e, Binding of full-length DmAgo1 to m7GTP-Sepharose in presence of increasing amounts of short RNA (23-mer). Input (20%) and bound fraction (20%) in assays were analyzed by western blotting using polyclonal anti-MBP (NEB) antibody.