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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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Readthrough efficiency for the sequences used for the GST reporter system. The lines in the center of the boxes indicate the median values; the box limits indicate the 25th and 75th percentiles calculated with R software; the whiskers extend to the minimum and maximum values; data points are plotted as open circles. n = 6 sample points. [psi−] values are indicated in white boxes, [PSI+] values are indicated in gray boxes.
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Figure 1: Readthrough efficiency for the sequences used for the GST reporter system. The lines in the center of the boxes indicate the median values; the box limits indicate the 25th and 75th percentiles calculated with R software; the whiskers extend to the minimum and maximum values; data points are plotted as open circles. n = 6 sample points. [psi−] values are indicated in white boxes, [PSI+] values are indicated in gray boxes.

Mentions: The second major potential drawback concerns the efficiency of stop codon readthrough. Indeed, the efficiency of unprogrammed stop codon readthrough is generally very low (< 0.3%) in yeast (10). We increased this efficiency by using a strain carrying the [PSI+] prion, which corresponds to the aggregated form of eukaryotic release factor 3 (eRF3). The sequestration of eRF3 in [PSI+] aggregates impairs the termination activity of eRF3, thereby increasing the efficiency of stop codon suppression (39). However, this increase is not sufficient to attain readthrough levels sufficiently high for the production of large amounts of GST. We previously identified a consensus readthrough motif (CA(A/G) N(U/C/G)A) that increased stop codon readthrough efficiency when located immediately downstream from the stop codon (10). In Saccharomyces cerevisiae, at least three genes (SFB2, TIP41, IXR1) naturally harbor this motif downstream from their stop codon. We first checked that these sequences actually promoted high levels of readthrough in [psi−] and [PSI+] strain, when used in a dual reporter system (Figure 1).


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)

Readthrough efficiency for the sequences used for the GST reporter system. The lines in the center of the boxes indicate the median values; the box limits indicate the 25th and 75th percentiles calculated with R software; the whiskers extend to the minimum and maximum values; data points are plotted as open circles. n = 6 sample points. [psi−] values are indicated in white boxes, [PSI+] values are indicated in gray boxes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150775&req=5

Figure 1: Readthrough efficiency for the sequences used for the GST reporter system. The lines in the center of the boxes indicate the median values; the box limits indicate the 25th and 75th percentiles calculated with R software; the whiskers extend to the minimum and maximum values; data points are plotted as open circles. n = 6 sample points. [psi−] values are indicated in white boxes, [PSI+] values are indicated in gray boxes.
Mentions: The second major potential drawback concerns the efficiency of stop codon readthrough. Indeed, the efficiency of unprogrammed stop codon readthrough is generally very low (< 0.3%) in yeast (10). We increased this efficiency by using a strain carrying the [PSI+] prion, which corresponds to the aggregated form of eukaryotic release factor 3 (eRF3). The sequestration of eRF3 in [PSI+] aggregates impairs the termination activity of eRF3, thereby increasing the efficiency of stop codon suppression (39). However, this increase is not sufficient to attain readthrough levels sufficiently high for the production of large amounts of GST. We previously identified a consensus readthrough motif (CA(A/G) N(U/C/G)A) that increased stop codon readthrough efficiency when located immediately downstream from the stop codon (10). In Saccharomyces cerevisiae, at least three genes (SFB2, TIP41, IXR1) naturally harbor this motif downstream from their stop codon. We first checked that these sequences actually promoted high levels of readthrough in [psi−] and [PSI+] strain, when used in a dual reporter system (Figure 1).

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