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Genetic analysis of L123 of the tRNA-mimicking eukaryote release factor eRF1, an amino acid residue critical for discrimination of stop codons.

Saito K, Ito K - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-city, Chiba 277-8562, Japan.

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Readthrough assay of Sc-eRF1 wild type (WT) and L123 mutants with paromomycin. Readthrough frequencies of Sc-eRF1 WT and L123 mutants in the presence of paromomycin (10 mg/ml). The assay strains, SKY81, SKY82, SKY83 and SKY84, were transformed with the expression vector, either of the wild type or position-123 mutants, of Sc-eRF1 on the p415GPD (LEU2 marker; Supplementary Table S6, rows ‘+paromomycin’). Paromomycin was added to the medium at the timing of temperature shift and refreshing. Wild type and mutant Sc-eRF1 readthrough frequencies (Supplementary Table S6) are indicated by percentage, where 100% readthrough was based on the results of a UGG reporter gene; values are indicated as mean ± SD from three independent measurements. Fold differences between selected stop codons, rounded to the first decimal place, are indicated over the bars with the values in the absence of paromomycin in the parentheses below (Supplementary Table S6).
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Figure 5: Readthrough assay of Sc-eRF1 wild type (WT) and L123 mutants with paromomycin. Readthrough frequencies of Sc-eRF1 WT and L123 mutants in the presence of paromomycin (10 mg/ml). The assay strains, SKY81, SKY82, SKY83 and SKY84, were transformed with the expression vector, either of the wild type or position-123 mutants, of Sc-eRF1 on the p415GPD (LEU2 marker; Supplementary Table S6, rows ‘+paromomycin’). Paromomycin was added to the medium at the timing of temperature shift and refreshing. Wild type and mutant Sc-eRF1 readthrough frequencies (Supplementary Table S6) are indicated by percentage, where 100% readthrough was based on the results of a UGG reporter gene; values are indicated as mean ± SD from three independent measurements. Fold differences between selected stop codons, rounded to the first decimal place, are indicated over the bars with the values in the absence of paromomycin in the parentheses below (Supplementary Table S6).

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).


Genetic analysis of L123 of the tRNA-mimicking eukaryote release factor eRF1, an amino acid residue critical for discrimination of stop codons.

Saito K, Ito K - Nucleic Acids Res. (2015)

Readthrough assay of Sc-eRF1 wild type (WT) and L123 mutants with paromomycin. Readthrough frequencies of Sc-eRF1 WT and L123 mutants in the presence of paromomycin (10 mg/ml). The assay strains, SKY81, SKY82, SKY83 and SKY84, were transformed with the expression vector, either of the wild type or position-123 mutants, of Sc-eRF1 on the p415GPD (LEU2 marker; Supplementary Table S6, rows ‘+paromomycin’). Paromomycin was added to the medium at the timing of temperature shift and refreshing. Wild type and mutant Sc-eRF1 readthrough frequencies (Supplementary Table S6) are indicated by percentage, where 100% readthrough was based on the results of a UGG reporter gene; values are indicated as mean ± SD from three independent measurements. Fold differences between selected stop codons, rounded to the first decimal place, are indicated over the bars with the values in the absence of paromomycin in the parentheses below (Supplementary Table S6).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Readthrough assay of Sc-eRF1 wild type (WT) and L123 mutants with paromomycin. Readthrough frequencies of Sc-eRF1 WT and L123 mutants in the presence of paromomycin (10 mg/ml). The assay strains, SKY81, SKY82, SKY83 and SKY84, were transformed with the expression vector, either of the wild type or position-123 mutants, of Sc-eRF1 on the p415GPD (LEU2 marker; Supplementary Table S6, rows ‘+paromomycin’). Paromomycin was added to the medium at the timing of temperature shift and refreshing. Wild type and mutant Sc-eRF1 readthrough frequencies (Supplementary Table S6) are indicated by percentage, where 100% readthrough was based on the results of a UGG reporter gene; values are indicated as mean ± SD from three independent measurements. Fold differences between selected stop codons, rounded to the first decimal place, are indicated over the bars with the values in the absence of paromomycin in the parentheses below (Supplementary Table S6).
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).

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.

View Article: PubMed Central - PubMed

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