Genetic analysis of L123 of the tRNA-mimicking eukaryote release factor eRF1, an amino acid residue critical for discrimination of stop codons.
Bottom Line: 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.
Affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-city, Chiba 277-8562, Japan.Show MeSH
Related in: MedlinePlus
Mentions: The ratio of readthrough frequency for UAA- and UAG-specific eRF1s, L123I and L123V, became less marked in the presence of paromomycin (Figure 5); e.g. for Sc-eRF1 L123I, UGA/UAA ratio was 2.6 (50.7/19.8%) in the presence of paromomycin, but was 3.7 in the absence of paromomycin. This might, in part, reflect specific inhibition of the eRF3-coupled decoding function of the position-123 residue by paromomycin. However, upon addition of paromomycin, the readthrough frequency in wild-type Sc-eRF1 transformants increased equally for all three stop codons (Figure 5 and Supplementary Table S6), i.e. paromomycin raised the basal levels of readthrough efficiency about 4-fold. Thus, the codon specificity ratios between the data sets in the presence and absence of paromomycin might not simply be applicable to the analysis of Hs-eRF1 L126 mutants with different eRF3s (Figure 3).
Affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-city, Chiba 277-8562, Japan.