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New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae.

Blanchet S, Cornu D, Argentini M, Namy O - Nucleic Acids Res. (2014)

Bottom Line: We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon.We also found that two different glutamine tRNA(Gln) were used to insert glutamine at UAA and UAG codons.This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, UMR8621, 91400 Orsay, France.

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Relative quantification of readthrough peptides. (A)–(D) MS extracted-ion chromatograms (XIC) of readthrough peptides in the IXR1 and SFB contexts. The X- and Y-axis correspond to LC elution time and absolute MS signal intensity, respectively. (E) Relative frequencies of the readthrough amino acids incorporated at UAA, UAG and UGA stop codons in the IXR1 and SFB2 contexts. Quantification data were processed and adjusted as described in the Materials and Methods.
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Figure 3: Relative quantification of readthrough peptides. (A)–(D) MS extracted-ion chromatograms (XIC) of readthrough peptides in the IXR1 and SFB contexts. The X- and Y-axis correspond to LC elution time and absolute MS signal intensity, respectively. (E) Relative frequencies of the readthrough amino acids incorporated at UAA, UAG and UGA stop codons in the IXR1 and SFB2 contexts. Quantification data were processed and adjusted as described in the Materials and Methods.

Mentions: In the IXR1 context, the same amino acids were found to be incorporated at the UAA and UAG stops, but their relative proportions differed between the two types of stop codon (Figure 3A and B). Indeed, tyrosine and glutamine residues were incorporated at similar frequencies at UAA stop codons (54 and 44%, respectively), whereas lysine was incorporated much less frequently (Figure 3A and E). In contrast, tyrosine was the main amino acid incorporated (92%) at the UAG stop codon, whereas glutamine and lysine residues were incorporated at similar, low frequencies (5% and 3%, respectively) (Figure 3B and E). In the TIP41 context, at the UAA stop codon, tyrosine and glutamine residues were incorporated at similar frequencies (54% and 42%) whereas lysine was incorporated less frequently (4%). While readthrough efficiency of the TIP41-UAA context was significantly lower than that of the IXR1-UAA context (Figure 1), incorporation efficiency of readthrough aminoacids was almost identical for both contexts (Supplementary Figures S3 and Figure 3A). At the UGA stop codon in the IXR1 context, tryptophan was the main amino acid inserted (82%), followed by cysteine (14%) and then arginine (Figure 3C and E). Very similar results were obtained for the UGA stop codon in the SFB2 context (Figure 3D and E).


New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae.

Blanchet S, Cornu D, Argentini M, Namy O - Nucleic Acids Res. (2014)

Relative quantification of readthrough peptides. (A)–(D) MS extracted-ion chromatograms (XIC) of readthrough peptides in the IXR1 and SFB contexts. The X- and Y-axis correspond to LC elution time and absolute MS signal intensity, respectively. (E) Relative frequencies of the readthrough amino acids incorporated at UAA, UAG and UGA stop codons in the IXR1 and SFB2 contexts. Quantification data were processed and adjusted as described in the Materials and Methods.
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Related In: Results  -  Collection

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Figure 3: Relative quantification of readthrough peptides. (A)–(D) MS extracted-ion chromatograms (XIC) of readthrough peptides in the IXR1 and SFB contexts. The X- and Y-axis correspond to LC elution time and absolute MS signal intensity, respectively. (E) Relative frequencies of the readthrough amino acids incorporated at UAA, UAG and UGA stop codons in the IXR1 and SFB2 contexts. Quantification data were processed and adjusted as described in the Materials and Methods.
Mentions: In the IXR1 context, the same amino acids were found to be incorporated at the UAA and UAG stops, but their relative proportions differed between the two types of stop codon (Figure 3A and B). Indeed, tyrosine and glutamine residues were incorporated at similar frequencies at UAA stop codons (54 and 44%, respectively), whereas lysine was incorporated much less frequently (Figure 3A and E). In contrast, tyrosine was the main amino acid incorporated (92%) at the UAG stop codon, whereas glutamine and lysine residues were incorporated at similar, low frequencies (5% and 3%, respectively) (Figure 3B and E). In the TIP41 context, at the UAA stop codon, tyrosine and glutamine residues were incorporated at similar frequencies (54% and 42%) whereas lysine was incorporated less frequently (4%). While readthrough efficiency of the TIP41-UAA context was significantly lower than that of the IXR1-UAA context (Figure 1), incorporation efficiency of readthrough aminoacids was almost identical for both contexts (Supplementary Figures S3 and Figure 3A). At the UGA stop codon in the IXR1 context, tryptophan was the main amino acid inserted (82%), followed by cysteine (14%) and then arginine (Figure 3C and E). Very similar results were obtained for the UGA stop codon in the SFB2 context (Figure 3D and E).

Bottom Line: We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon.We also found that two different glutamine tRNA(Gln) were used to insert glutamine at UAA and UAG codons.This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, UMR8621, 91400 Orsay, France.

Show MeSH