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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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Domains and sequence alignment around L123 of Sc-eRF1. (A) Schematic drawing of the domain structure of eRF1. Three domains, according to the structure of eRF1 (domains N, M and C) are shown with the amino acid residue numbers at the domain junctions. Relevant motifs and sites mentioned in this manuscript are also indicated. (B) Alignment of eRF1 around the position-123 amino acid residue. Amino acid numbering is based on that of Saccharomyces cerevisiae eRF1. Position-123 is shown in red. The YxCxxxF motif is indicated at the bottom. Species with standard and variant stop codon specificity are shown in orange for UAA, UAG and UGA, blue for UAA and UAG and green for UGA. Amino acids at position-123 are highly conserved within each of those categories. The codon specificities of eRF1 indicated here with asterisks (*) have been reported previously (9,27–29) and others are proposed from their codon usage analysis (11,19).
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Figure 1: Domains and sequence alignment around L123 of Sc-eRF1. (A) Schematic drawing of the domain structure of eRF1. Three domains, according to the structure of eRF1 (domains N, M and C) are shown with the amino acid residue numbers at the domain junctions. Relevant motifs and sites mentioned in this manuscript are also indicated. (B) Alignment of eRF1 around the position-123 amino acid residue. Amino acid numbering is based on that of Saccharomyces cerevisiae eRF1. Position-123 is shown in red. The YxCxxxF motif is indicated at the bottom. Species with standard and variant stop codon specificity are shown in orange for UAA, UAG and UGA, blue for UAA and UAG and green for UGA. Amino acids at position-123 are highly conserved within each of those categories. The codon specificities of eRF1 indicated here with asterisks (*) have been reported previously (9,27–29) and others are proposed from their codon usage analysis (11,19).

Mentions: In eukaryotes, the class 1 RF, eRF1, and the class 2 RF, eRF3, are distinct from eubacterial RFs. eRF1 (encoded by SUP45 in budding yeast) recognizes all three stop codons, i.e. has ‘omnipotent’ recognition, and stimulates hydrolysis of peptidyl tRNA by the GGQ motif (6). eRF1 has three structural domains (Figure 1A) (7). Domain N structurally corresponds to the anticodon stem-loop of tRNA and has been shown to participate in omnipotent stop codon recognition (8). Domain N contains crucial motifs for stop codon discrimination, such as TASNIKS and YxCxxxF (9,10). Domain M contains the universal GGQ motif at the tip of the domain, which is comparable to the CCA terminal of tRNA. Domain C contains the principal site for interaction with eRF3, named ‘site 1’. On the other hand, eRF3 (encoded by SUP35 in budding yeast) shares high homology with the translational GTPase eEF1A/EF-Tu subfamily (11). Unlike RF3, eRF3 forms a heterodimer complex with eRF1, preferably in the presence of GTP (eRF1–eRF3–GTP complex), prior to entering the ribosomal A site (12), and stimulates peptide release for decoding of stop codons (13,14). This strongly suggests that it is functionally similar to the tRNA–eEF1A–GTP complex for decoding of sense codons.


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)

Domains and sequence alignment around L123 of Sc-eRF1. (A) Schematic drawing of the domain structure of eRF1. Three domains, according to the structure of eRF1 (domains N, M and C) are shown with the amino acid residue numbers at the domain junctions. Relevant motifs and sites mentioned in this manuscript are also indicated. (B) Alignment of eRF1 around the position-123 amino acid residue. Amino acid numbering is based on that of Saccharomyces cerevisiae eRF1. Position-123 is shown in red. The YxCxxxF motif is indicated at the bottom. Species with standard and variant stop codon specificity are shown in orange for UAA, UAG and UGA, blue for UAA and UAG and green for UGA. Amino acids at position-123 are highly conserved within each of those categories. The codon specificities of eRF1 indicated here with asterisks (*) have been reported previously (9,27–29) and others are proposed from their codon usage analysis (11,19).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Domains and sequence alignment around L123 of Sc-eRF1. (A) Schematic drawing of the domain structure of eRF1. Three domains, according to the structure of eRF1 (domains N, M and C) are shown with the amino acid residue numbers at the domain junctions. Relevant motifs and sites mentioned in this manuscript are also indicated. (B) Alignment of eRF1 around the position-123 amino acid residue. Amino acid numbering is based on that of Saccharomyces cerevisiae eRF1. Position-123 is shown in red. The YxCxxxF motif is indicated at the bottom. Species with standard and variant stop codon specificity are shown in orange for UAA, UAG and UGA, blue for UAA and UAG and green for UGA. Amino acids at position-123 are highly conserved within each of those categories. The codon specificities of eRF1 indicated here with asterisks (*) have been reported previously (9,27–29) and others are proposed from their codon usage analysis (11,19).
Mentions: In eukaryotes, the class 1 RF, eRF1, and the class 2 RF, eRF3, are distinct from eubacterial RFs. eRF1 (encoded by SUP45 in budding yeast) recognizes all three stop codons, i.e. has ‘omnipotent’ recognition, and stimulates hydrolysis of peptidyl tRNA by the GGQ motif (6). eRF1 has three structural domains (Figure 1A) (7). Domain N structurally corresponds to the anticodon stem-loop of tRNA and has been shown to participate in omnipotent stop codon recognition (8). Domain N contains crucial motifs for stop codon discrimination, such as TASNIKS and YxCxxxF (9,10). Domain M contains the universal GGQ motif at the tip of the domain, which is comparable to the CCA terminal of tRNA. Domain C contains the principal site for interaction with eRF3, named ‘site 1’. On the other hand, eRF3 (encoded by SUP35 in budding yeast) shares high homology with the translational GTPase eEF1A/EF-Tu subfamily (11). Unlike RF3, eRF3 forms a heterodimer complex with eRF1, preferably in the presence of GTP (eRF1–eRF3–GTP complex), prior to entering the ribosomal A site (12), and stimulates peptide release for decoding of stop codons (13,14). This strongly suggests that it is functionally similar to the tRNA–eEF1A–GTP complex for decoding of sense 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.

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