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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 Hs-eRF1 mutants with Sc- or Hs-eRF3. (A) Readthrough frequencies of Hs-eRF1 wild type (WT) and position-123 mutants (L126 in human) with Sc-eRF3. The assay strains, S13-I01, S13-I03, S13-I05 and S13-I07, were transformed with the expression vector, either of wild type or position-123 mutants, of Hs-eRF1 on the p416GPD (URA3 marker) (Supplementary Table S4, rows ‘Sc-eRF3’). (B) Readthrough frequencies of Hs-eRF1 wild type (WT) and position-123 mutants (L126 in human) with Hs-eRF3c. The assay strains, SKY106, SKY107, SKY108 and SKY109, were transformed with the expression vector, either of wild type or position-123 mutants of Hs-eRF1 on the p415GPD (LEU2 marker) as well as with the expression vector of Hs-eRF3c on p416GPD (URA3 marker) (Supplementary Table S4, rows ‘Hs-eRF3c’). (C) Readthrough frequencies of Hs-eRF1 mutants of putative stop codon-binding residues (T32, E55 V71, Y125 and C127 of eRF1 in human numbering) with Sc-eRF3. The assay strains, S13-I01, S13-I03, S13-I05 and S13-I07, were transformed with the expression vector, either of wild type or mutants of Hs-eRF1 on p416GPD (URA3 marker) (Supplementary Table S5, rows ‘Sc-eRF3’; see Figure 3A and Supplementary Table S4 for wild type Hs-eRF1). (D) Readthrough frequencies of Hs-eRF1 mutants of putative stop codon-binding residues (T32, E55 V71, Y125 and C127 of eRF1 in human numbering) with Hs-eRF3c. The assay strains, SKY106, SKY107, SKY108 and SKY109, were transformed with the expression vector, either of wild type or mutants of Hs-eRF1 on p415GPD (LEU2 marker), as well as with the expression vector of Hs-eRF3c on p416GPD (URA3 marker) (Supplementary Table S5, rows ‘Hs-eRF3c’; see Figure 3B and Supplementary Table S4 for wild type Hs-eRF1). Readthrough frequencies are indicated as percentages, 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 (Supplementary Tables S4 and S5), are indicated over the bars.
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Figure 3: Readthrough assay of Hs-eRF1 mutants with Sc- or Hs-eRF3. (A) Readthrough frequencies of Hs-eRF1 wild type (WT) and position-123 mutants (L126 in human) with Sc-eRF3. The assay strains, S13-I01, S13-I03, S13-I05 and S13-I07, were transformed with the expression vector, either of wild type or position-123 mutants, of Hs-eRF1 on the p416GPD (URA3 marker) (Supplementary Table S4, rows ‘Sc-eRF3’). (B) Readthrough frequencies of Hs-eRF1 wild type (WT) and position-123 mutants (L126 in human) with Hs-eRF3c. The assay strains, SKY106, SKY107, SKY108 and SKY109, were transformed with the expression vector, either of wild type or position-123 mutants of Hs-eRF1 on the p415GPD (LEU2 marker) as well as with the expression vector of Hs-eRF3c on p416GPD (URA3 marker) (Supplementary Table S4, rows ‘Hs-eRF3c’). (C) Readthrough frequencies of Hs-eRF1 mutants of putative stop codon-binding residues (T32, E55 V71, Y125 and C127 of eRF1 in human numbering) with Sc-eRF3. The assay strains, S13-I01, S13-I03, S13-I05 and S13-I07, were transformed with the expression vector, either of wild type or mutants of Hs-eRF1 on p416GPD (URA3 marker) (Supplementary Table S5, rows ‘Sc-eRF3’; see Figure 3A and Supplementary Table S4 for wild type Hs-eRF1). (D) Readthrough frequencies of Hs-eRF1 mutants of putative stop codon-binding residues (T32, E55 V71, Y125 and C127 of eRF1 in human numbering) with Hs-eRF3c. The assay strains, SKY106, SKY107, SKY108 and SKY109, were transformed with the expression vector, either of wild type or mutants of Hs-eRF1 on p415GPD (LEU2 marker), as well as with the expression vector of Hs-eRF3c on p416GPD (URA3 marker) (Supplementary Table S5, rows ‘Hs-eRF3c’; see Figure 3B and Supplementary Table S4 for wild type Hs-eRF1). Readthrough frequencies are indicated as percentages, 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 (Supplementary Tables S4 and S5), are indicated over the bars.

Mentions: To test phylogenetic functional conservation of the mutational effects of the position-123 residue, human eRF1 (Hs-eRF1) was mutated at the same position, residue L126 (in human numbering, which corresponds to L123 of S. cerevisiae eRF1), and applied to the readthrough assay. As shown in Figure 3A and Supplementary Table S4, most of the Hs-eRF1 L126 mutants exhibited similar codon-specificity patterns to the corresponding L123 mutants of Sc-eRF1, although less markedly so.


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 Hs-eRF1 mutants with Sc- or Hs-eRF3. (A) Readthrough frequencies of Hs-eRF1 wild type (WT) and position-123 mutants (L126 in human) with Sc-eRF3. The assay strains, S13-I01, S13-I03, S13-I05 and S13-I07, were transformed with the expression vector, either of wild type or position-123 mutants, of Hs-eRF1 on the p416GPD (URA3 marker) (Supplementary Table S4, rows ‘Sc-eRF3’). (B) Readthrough frequencies of Hs-eRF1 wild type (WT) and position-123 mutants (L126 in human) with Hs-eRF3c. The assay strains, SKY106, SKY107, SKY108 and SKY109, were transformed with the expression vector, either of wild type or position-123 mutants of Hs-eRF1 on the p415GPD (LEU2 marker) as well as with the expression vector of Hs-eRF3c on p416GPD (URA3 marker) (Supplementary Table S4, rows ‘Hs-eRF3c’). (C) Readthrough frequencies of Hs-eRF1 mutants of putative stop codon-binding residues (T32, E55 V71, Y125 and C127 of eRF1 in human numbering) with Sc-eRF3. The assay strains, S13-I01, S13-I03, S13-I05 and S13-I07, were transformed with the expression vector, either of wild type or mutants of Hs-eRF1 on p416GPD (URA3 marker) (Supplementary Table S5, rows ‘Sc-eRF3’; see Figure 3A and Supplementary Table S4 for wild type Hs-eRF1). (D) Readthrough frequencies of Hs-eRF1 mutants of putative stop codon-binding residues (T32, E55 V71, Y125 and C127 of eRF1 in human numbering) with Hs-eRF3c. The assay strains, SKY106, SKY107, SKY108 and SKY109, were transformed with the expression vector, either of wild type or mutants of Hs-eRF1 on p415GPD (LEU2 marker), as well as with the expression vector of Hs-eRF3c on p416GPD (URA3 marker) (Supplementary Table S5, rows ‘Hs-eRF3c’; see Figure 3B and Supplementary Table S4 for wild type Hs-eRF1). Readthrough frequencies are indicated as percentages, 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 (Supplementary Tables S4 and S5), are indicated over the bars.
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Figure 3: Readthrough assay of Hs-eRF1 mutants with Sc- or Hs-eRF3. (A) Readthrough frequencies of Hs-eRF1 wild type (WT) and position-123 mutants (L126 in human) with Sc-eRF3. The assay strains, S13-I01, S13-I03, S13-I05 and S13-I07, were transformed with the expression vector, either of wild type or position-123 mutants, of Hs-eRF1 on the p416GPD (URA3 marker) (Supplementary Table S4, rows ‘Sc-eRF3’). (B) Readthrough frequencies of Hs-eRF1 wild type (WT) and position-123 mutants (L126 in human) with Hs-eRF3c. The assay strains, SKY106, SKY107, SKY108 and SKY109, were transformed with the expression vector, either of wild type or position-123 mutants of Hs-eRF1 on the p415GPD (LEU2 marker) as well as with the expression vector of Hs-eRF3c on p416GPD (URA3 marker) (Supplementary Table S4, rows ‘Hs-eRF3c’). (C) Readthrough frequencies of Hs-eRF1 mutants of putative stop codon-binding residues (T32, E55 V71, Y125 and C127 of eRF1 in human numbering) with Sc-eRF3. The assay strains, S13-I01, S13-I03, S13-I05 and S13-I07, were transformed with the expression vector, either of wild type or mutants of Hs-eRF1 on p416GPD (URA3 marker) (Supplementary Table S5, rows ‘Sc-eRF3’; see Figure 3A and Supplementary Table S4 for wild type Hs-eRF1). (D) Readthrough frequencies of Hs-eRF1 mutants of putative stop codon-binding residues (T32, E55 V71, Y125 and C127 of eRF1 in human numbering) with Hs-eRF3c. The assay strains, SKY106, SKY107, SKY108 and SKY109, were transformed with the expression vector, either of wild type or mutants of Hs-eRF1 on p415GPD (LEU2 marker), as well as with the expression vector of Hs-eRF3c on p416GPD (URA3 marker) (Supplementary Table S5, rows ‘Hs-eRF3c’; see Figure 3B and Supplementary Table S4 for wild type Hs-eRF1). Readthrough frequencies are indicated as percentages, 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 (Supplementary Tables S4 and S5), are indicated over the bars.
Mentions: To test phylogenetic functional conservation of the mutational effects of the position-123 residue, human eRF1 (Hs-eRF1) was mutated at the same position, residue L126 (in human numbering, which corresponds to L123 of S. cerevisiae eRF1), and applied to the readthrough assay. As shown in Figure 3A and Supplementary Table S4, most of the Hs-eRF1 L126 mutants exhibited similar codon-specificity patterns to the corresponding L123 mutants of Sc-eRF1, although less markedly so.

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