title: Genetic analysis of L123 of the tRNA-mimicking eukaryote release factor eRF1, an amino acid residue critical for discrimination of stop codons.
abstract: In eukaryotes, the tRNA-mimicking polypeptide-chain release factor, eRF1, decodes stop codons on the ribosome in a complex with eRF3; this complex exhibits striking structural similarity to the tRNA-eEF1A-GTP complex. Although amino acid residues or motifs of eRF1 that are critical for stop codon discrimination have been identified, the details of the molecular mechanisms involved in the function of the ribosomal decoding site remain obscure. Here, we report analyses of the position-123 amino acid of eRF1 (L123 in Saccharomyces cerevisiae eRF1), a residue that is phylogenetically conserved among species with canonical and variant genetic codes. In vivo readthrough efficiency analysis and genetic growth complementation analysis of the residue-123 systematic mutants suggested that this amino acid functions in stop codon discrimination in a manner coupled with eRF3 binding, and distinctive from previously reported adjacent residues. Furthermore, aminoglycoside antibiotic sensitivity analysis and ribosomal docking modeling of eRF1 in a quasi-A/T state suggested a functional interaction between the side chain of L123 and ribosomal residues critical for codon recognition in the decoding site, as a molecular explanation for coupling with eRF3. Our results provide insights into the molecular mechanisms underlying stop codon discrimination by a tRNA-mimicking protein on the ribosome.
authors.affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-city, Chiba 277-8562, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Saito
authors.affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-city, Chiba 277-8562, Japan itokoichi@k.u-tokyo.ac.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Ito
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D018388:Codon, Terminator; D007930:Leucine / Q000502:physiology*; D009154:Mutation; D010303:Paromomycin / Q000494:pharmacology; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D018388:Codon, Terminator; C489002:ETF1 protein, human; D010454:Peptide Termination Factors; C080775:SUP45 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D010303:Paromomycin; D007930:Leucine