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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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Alanine insertion at stop codons after the coexpression of IXR1-UAA and IXR1-UAG readthrough proteins with modified 3′GUU5′ and 3′GUC5′ tRNAsAla. MS extracted-ion chromatograms (XIC) of readthrough peptides with lysine, glutamine, tyrosine and alanine incorporated at the UAA and UAG stop codons are shown in left panels. To better visualize alanine incorporation at UAA and UAG stop codons, a zoom of the elution region is shown on the right side of each XIC. The alanine incorporated readthrough peptide (KNEAQINNLSPILGYW) is detected only when IXR1-UAA and IXR1-UAG are coexpressed with tRNAAla3′GUU5′ (panel A) or tRNAAla3′GUC5′ (panel D), respectively. The asterisk (*) indicates a peak corresponding to the alanine isobaric peptide with alanine incorporated at the glutamine site downstream from the stop codon (KNEQAINNLSPILGYW).
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Figure 4: Alanine insertion at stop codons after the coexpression of IXR1-UAA and IXR1-UAG readthrough proteins with modified 3′GUU5′ and 3′GUC5′ tRNAsAla. MS extracted-ion chromatograms (XIC) of readthrough peptides with lysine, glutamine, tyrosine and alanine incorporated at the UAA and UAG stop codons are shown in left panels. To better visualize alanine incorporation at UAA and UAG stop codons, a zoom of the elution region is shown on the right side of each XIC. The alanine incorporated readthrough peptide (KNEAQINNLSPILGYW) is detected only when IXR1-UAA and IXR1-UAG are coexpressed with tRNAAla3′GUU5′ (panel A) or tRNAAla3′GUC5′ (panel D), respectively. The asterisk (*) indicates a peak corresponding to the alanine isobaric peptide with alanine incorporated at the glutamine site downstream from the stop codon (KNEQAINNLSPILGYW).

Mentions: The modified tRNAs were coexpressed with readthrough GST in the [PSI+] ΔUPF1 yeast strain. LC-MS/MS analyses were performed and showed that alanine was incorporated at the UAA codon only if the tRNAAla carried the 3′GUU5′ anticodon (Figure 4A and B and Supplementary Figure S4a) Thus, this tRNAAla can decode UAA codons only if it carries this anticodon. At the UAG codon, alanine was detected only in the presence of the modified tRNAAla3′GUC5′ (Figure 4C and D and Supplementary Figure S4b). These results clearly demonstrate that anticodon identity is the principal determinant of stop codon decoding and that the observed differences in glutamine incorporation are due to differences in the relative abundance of tRNAs able to decode the various stop codons.


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)

Alanine insertion at stop codons after the coexpression of IXR1-UAA and IXR1-UAG readthrough proteins with modified 3′GUU5′ and 3′GUC5′ tRNAsAla. MS extracted-ion chromatograms (XIC) of readthrough peptides with lysine, glutamine, tyrosine and alanine incorporated at the UAA and UAG stop codons are shown in left panels. To better visualize alanine incorporation at UAA and UAG stop codons, a zoom of the elution region is shown on the right side of each XIC. The alanine incorporated readthrough peptide (KNEAQINNLSPILGYW) is detected only when IXR1-UAA and IXR1-UAG are coexpressed with tRNAAla3′GUU5′ (panel A) or tRNAAla3′GUC5′ (panel D), respectively. The asterisk (*) indicates a peak corresponding to the alanine isobaric peptide with alanine incorporated at the glutamine site downstream from the stop codon (KNEQAINNLSPILGYW).
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Related In: Results  -  Collection

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Figure 4: Alanine insertion at stop codons after the coexpression of IXR1-UAA and IXR1-UAG readthrough proteins with modified 3′GUU5′ and 3′GUC5′ tRNAsAla. MS extracted-ion chromatograms (XIC) of readthrough peptides with lysine, glutamine, tyrosine and alanine incorporated at the UAA and UAG stop codons are shown in left panels. To better visualize alanine incorporation at UAA and UAG stop codons, a zoom of the elution region is shown on the right side of each XIC. The alanine incorporated readthrough peptide (KNEAQINNLSPILGYW) is detected only when IXR1-UAA and IXR1-UAG are coexpressed with tRNAAla3′GUU5′ (panel A) or tRNAAla3′GUC5′ (panel D), respectively. The asterisk (*) indicates a peak corresponding to the alanine isobaric peptide with alanine incorporated at the glutamine site downstream from the stop codon (KNEQAINNLSPILGYW).
Mentions: The modified tRNAs were coexpressed with readthrough GST in the [PSI+] ΔUPF1 yeast strain. LC-MS/MS analyses were performed and showed that alanine was incorporated at the UAA codon only if the tRNAAla carried the 3′GUU5′ anticodon (Figure 4A and B and Supplementary Figure S4a) Thus, this tRNAAla can decode UAA codons only if it carries this anticodon. At the UAG codon, alanine was detected only in the presence of the modified tRNAAla3′GUC5′ (Figure 4C and D and Supplementary Figure S4b). These results clearly demonstrate that anticodon identity is the principal determinant of stop codon decoding and that the observed differences in glutamine incorporation are due to differences in the relative abundance of tRNAs able to decode the various stop codons.

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