New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae.
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.
Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, UMR8621, 91400 Orsay, France.Show MeSH
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.
Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, UMR8621, 91400 Orsay, France.