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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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Purification and verification of readthrough GST proteins. (A) The chromatograms show absorbance at 280 nm. The red curve corresponds to in-frame GST purification; the blue curve corresponds to GST-IXRI-TAG purification. The elution peaks appears in fraction 3. (B) SDS-PAGE gel stained by Coomassie Blue. Lane 1: whole-cell extract; lane 2: flowthrough after binding of the extract; lane 3: in-frame GST, and lane 4: purified readthrough protein, GST-IXRI-TAG. For lanes 3 and 4, 1.8 μg of protein was loaded on the gel. (C) The presence of the GST protein was checked by western blotting with an antibody against GST. Lane 1 corresponds to the whole-cell extract, lane 2 corresponds to the flowthrough after binding of the extract, lane 3 to in-frame GST, and lane 4 to purified readthrough protein, GST-IXRI-TAA. For lanes 3 and 4, 0.5 μg of protein was loaded on the gel.
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Figure 2: Purification and verification of readthrough GST proteins. (A) The chromatograms show absorbance at 280 nm. The red curve corresponds to in-frame GST purification; the blue curve corresponds to GST-IXRI-TAG purification. The elution peaks appears in fraction 3. (B) SDS-PAGE gel stained by Coomassie Blue. Lane 1: whole-cell extract; lane 2: flowthrough after binding of the extract; lane 3: in-frame GST, and lane 4: purified readthrough protein, GST-IXRI-TAG. For lanes 3 and 4, 1.8 μg of protein was loaded on the gel. (C) The presence of the GST protein was checked by western blotting with an antibody against GST. Lane 1 corresponds to the whole-cell extract, lane 2 corresponds to the flowthrough after binding of the extract, lane 3 to in-frame GST, and lane 4 to purified readthrough protein, GST-IXRI-TAA. For lanes 3 and 4, 0.5 μg of protein was loaded on the gel.

Mentions: The IXR1-TAG sequence was found to be the most efficient (25% of stop codon readthrough) and was therefore used in the GST purification protocol. Cell lysates were prepared as described in the Materials and Methods and GST proteins were purified on glutathione affinity columns. For GST-IXR1-TAG purification, a single peak was eluted from the column at the same position as the in-frame GST (Figure 2a). This peak corresponded to a single band at the position of the in-frame GST in the elution fractions, as revealed by SDS-PAGE and Coomassie Blue staining (Figure 2b). Using an antibody specific for GST, we confirmed that the single band observed was indeed GST (Figure 2c). These data demonstrate that this reporter system is suitable for the purification of readthrough GST proteins.


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)

Purification and verification of readthrough GST proteins. (A) The chromatograms show absorbance at 280 nm. The red curve corresponds to in-frame GST purification; the blue curve corresponds to GST-IXRI-TAG purification. The elution peaks appears in fraction 3. (B) SDS-PAGE gel stained by Coomassie Blue. Lane 1: whole-cell extract; lane 2: flowthrough after binding of the extract; lane 3: in-frame GST, and lane 4: purified readthrough protein, GST-IXRI-TAG. For lanes 3 and 4, 1.8 μg of protein was loaded on the gel. (C) The presence of the GST protein was checked by western blotting with an antibody against GST. Lane 1 corresponds to the whole-cell extract, lane 2 corresponds to the flowthrough after binding of the extract, lane 3 to in-frame GST, and lane 4 to purified readthrough protein, GST-IXRI-TAA. For lanes 3 and 4, 0.5 μg of protein was loaded on the gel.
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Related In: Results  -  Collection

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Figure 2: Purification and verification of readthrough GST proteins. (A) The chromatograms show absorbance at 280 nm. The red curve corresponds to in-frame GST purification; the blue curve corresponds to GST-IXRI-TAG purification. The elution peaks appears in fraction 3. (B) SDS-PAGE gel stained by Coomassie Blue. Lane 1: whole-cell extract; lane 2: flowthrough after binding of the extract; lane 3: in-frame GST, and lane 4: purified readthrough protein, GST-IXRI-TAG. For lanes 3 and 4, 1.8 μg of protein was loaded on the gel. (C) The presence of the GST protein was checked by western blotting with an antibody against GST. Lane 1 corresponds to the whole-cell extract, lane 2 corresponds to the flowthrough after binding of the extract, lane 3 to in-frame GST, and lane 4 to purified readthrough protein, GST-IXRI-TAA. For lanes 3 and 4, 0.5 μg of protein was loaded on the gel.
Mentions: The IXR1-TAG sequence was found to be the most efficient (25% of stop codon readthrough) and was therefore used in the GST purification protocol. Cell lysates were prepared as described in the Materials and Methods and GST proteins were purified on glutathione affinity columns. For GST-IXR1-TAG purification, a single peak was eluted from the column at the same position as the in-frame GST (Figure 2a). This peak corresponded to a single band at the position of the in-frame GST in the elution fractions, as revealed by SDS-PAGE and Coomassie Blue staining (Figure 2b). Using an antibody specific for GST, we confirmed that the single band observed was indeed GST (Figure 2c). These data demonstrate that this reporter system is suitable for the purification of readthrough GST proteins.

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