A role for aromatic amino acids in the binding of Xenopus ribosomal protein L5 to 5S rRNA.
The formation of the Xenopus L5-5S rRNA complex depends on nonelectrostatic interactions. Fluorescence assays with 1-anilino-8-naphthalenesulfonate demonstrate that a hydrophobic region on L5 becomes exposed upon removal of bound 5S rRNA by treatment with ribonucleases. Several conserved aromatic amino acids, mostly tyrosines, were identified by comparative sequence analysis and changed individually to alanine. Substitution with alanine at any of three positions, Y86, Y99, or Y226, essentially abolishes RNA-binding activity, whereas those made at Y95 and Y207 have more modest effects. Replacement with phenylalanine at Y86 and Y226 does not change binding affinity, indicating that the aromatic ring of the side chain, not the hydroxyl group, is the critical functionality for binding. Alternatively, the phenolic hydroxyls at Y99 and Y207 do contribute to binding. The structural integrity of the mutant proteins was assessed using thermal denaturation and limited digestion with proteases. The T(m) of Y99A is 10 degrees C lower than that of the wild-type protein, and there are some differences in the protease digestion patterns that together indicate the structure of this mutant has been significantly perturbed. The structures of the other variants are not detectably different from the wild-type protein. These results provide evidence that intermolecular stacking interactions involving at least two tyrosine residues, Y86 and Y226, are necessary for formation of the L5-5S rRNA complex and can account, at least in part, for the contribution nonelectrostatic interactions make to the free energy of binding.
DiNitto
J P
JP
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Huber
P W
PW
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D024322:Amino Acids, Aromatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002942:Circular Dichroism; D017124:Conserved Sequence; D010450:Endopeptidases / Q000378:metabolism; D006358:Hot Temperature; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D011489:Protein Denaturation; D012341:RNA, Ribosomal, 5S / Q000737:chemistry / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
D024322:Amino Acids, Aromatic; D012341:RNA, Ribosomal, 5S; D012269:Ribosomal Proteins; C029742:ribosomal protein L5; D014443:Tyrosine; D010450:Endopeptidases; D000409:Alanine