FIGURE 4.

Identification of amino acid substitutions at position 288 of FBF-2 that alter RNA specificity. (A) Yeast three-hybrid selection used to identify stacking amino acid residues in FBF-2 Repeat 3 with altered RNA specificities. Position 288 of FBF-2 was randomized, and the library of mutants was screened with RNAs substituted at base +7. (B) Specificity testing of yeast 3-hybrid selected FBF-2 mutants with four different RNAs. (Top left) Core elements of RNA sequences tested in the three-hybrid assay; (top right) diagram of RNA bases (ellipses) and stacking residues (dark gray rectangle) tested. RNAs with base substitutions (gray) at +7 were analyzed for binding with WT FBF-2 (R288) and mutants (W, F, and Y) in the three-hybrid assay. β-Galactosidase activity reflects strength of the interactions (Hook et al. 2005).