Xenopus ribosomal protein L5 was expressed in Escherichia coli and exhibits high affinity (K = 2 nM) and specificity for oocyte 5 S rRNA. The pH dependence of the association constant for the complex reveals an ionization with a pK value of 10.1, indicating that tyrosine and/or lysine residues are important for specific binding of L5 to the RNA. Formation of the L55 S rRNA complex is remarkably insensitive to ionic strength, providing evidence that nonelectrostatic interactions make significant contributions to binding. Together, these results suggest that one or more tyrosine residues may form critical contacts through stacking interactions with bases in the RNA. In order to locate recognition elements within 5 S rRNA, we measured binding of L5 to a collection of site-specific mutants. Mutations in the RNA that affected the interaction are confined to the hairpin structure comprised of helix III and loop C. Earlier experiments with a rhodium structural probe had shown that the two-nucleotide bulge in helix III and the intrinsic structure of loop C create sites in the major groove that are opened and accessible to stacking interactions with the metal complex. In the present studies, we detect a correlation between the intercalative binding of the rhodium complex to mutants in the hairpin and binding of L5, supporting the proposal that binding of the protein is mediated, in some part, by stacking interactions. Furthermore, the results from mutagenesis establish that, despite overlapping binding sites on 5 S rRNA, L5 and transcription factor IIIA utilize distinct structural elements for recognition.

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