Contributions of basic residues to ribosomal protein L11 recognition of RNA.
The C-terminal domain of ribosomal protein L11, L11-C76, binds in the distorted minor groove of a helix within a 58 nucleotide domain of 23 S rRNA. To study the electrostatic component of RNA recognition in this protein, arginine and lysine residues have been individually mutated to alanine or methionine residues at the nine sequence positions that are conserved as basic residues among bacterial L11 homologs. In measurements of the salt dependence of RNA-binding, five of these mutants have a reduced value of - partial differentiallog(K(obs))/ partial differentiallog[KCl] as compared to the parent L11-C76 sequence, indicating that these residues interact with the RNA electrostatic field. These five residues are located at the perimeter of the RNA-binding surface of the protein; all five of them form salt bridges with phosphates in the crystal structure of the complex. A sixth residue, Lys47, was found to make an electrostatic contribution to binding when measurements were made at pH 6.0, but not at pH 7.0; its pK in the free protein must be <6.5. The unusual behavior of Lys47 is explained by its burial in the hydrophobic core of the free protein, and unburial in the RNA-bound protein, where it forms a salt bridge with a phosphate. The contributions of these six residues to the electrostatic component of binding are not additive; thus the magnitude of the salt dependence cannot be used to count the number of ionic interactions in this complex. By interacting with irregular features of the RNA backbone, including an S-turn, these basic residues contribute to the specificity of L11 for its target site.
GuhaThakurta
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Department of Chemistry, Johns Hopkins University, Baltimore, MD, 21218, USA.
Draper
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D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D017510:Protein Folding; D012335:RNA, Ribosomal / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*
D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C027624:ribosomal protein L11