Figure 3.
Brat interaction with the Nos/Pum/NRE ternary complex in vitro. (A) The Brat NHL domain binds specifically to wild-type ternary complexes in vitro. The figure is a Western blot probed with anti-Xpress antibody to detect the tag present on both the recombinant His6–Nos and His6–Brat molecules. Lane 1 contains ∼5% of input protein for the experiments of lanes 2–5, and lane 6 contains the same amount of input protein (i.e., Brat only) for the experiments of lanes 7 and 8. After incubation and capture on glutathione agarose beads, bound proteins were eluted and visualized by Western blot. Approximately 1% of the input Brat is bound in the presence of wild-type components (lane 2). The mutant (mut) RNA in the reactions applied to lanes 3, 5, and 8 bears the AA17CC mutation, which specifically prevents Nos binding to the Pum/NRE complex (Sonoda and Wharton 1999). The G1330D mutant Pum (lanes 4,5) is described in the text. (B) Substitutions in the NHL domains of the Bratfs mutant proteins prevent binding to the Pum/Nos/NRE complex. The figure is a Western blot probed with α-CC to detect proteins in embryonic extracts (genotype indicated above) that bind to ternary Pum/Nos/NRE complexes assembled in vitro with purified components. The blot was reprobed with antibodies to α-tubulin, which serves as a loading control. Lanes 1–3 contain ∼5% of input protein, and the reaction loaded in lane 4 contained the NRE AA17CC mutant.