Quantitation of readthrough of termination codons in yeast using a novel gene fusion assay.
A simple quantitative in vivo assay has been developed for measuring the efficiency of translation of one or other of the three termination codons. UAA, UAG and UGA in Saccharomyces cerevisiae. The assay employs a 3-phosphoglycerate kinase-beta-galactosidase gene fusion, carried on a multicopy plasmid, in which the otherwise retained reading frame is disrupted by one or other of the three termination codons. Termination readthrough is thus quantitated by measuring beta-galactosidase in transformed strains. Using these plasmids to quantitate the endogenous levels of termination readthrough we show that readthrough of all three codons can be detected in a non-suppressor (sup+) strain of S. cerevisiae. The efficiency of this endogenous readthrough is much higher in a [psi+] strain than in a [psi-] strain with the UGA codon being the leakiest in the nucleotide context used. The utility of the assay plasmids for studying genetic modifiers of nonsense suppressors is also shown by their use to demonstrate that the cytoplasmic genetic determinant [psi+] broadens the decoding properties of a serine-inserting UAA suppressor tRNA (SUQ5) to allow it to translate the other two termination codons in the order of efficiency UAA greater than UAG greater than UGA.
Firoozan
M
M
Biological Laboratory, University of Kent, Canterbury, U.K.
Grant
C M
CM
Duarte
J A
JA
Tuite
M F
MF
D016375:Antisense Elements (Genetics); D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010443:Peptide Chain Termination, Translational; D010735:Phosphoglycerate Kinase / Q000096:biosynthesis; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D012362:RNA, Transfer, Ser / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013104:Spheroplasts; D013489:Suppression, Genetic; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis
D016375:Antisense Elements (Genetics); D003062:Codon; D012362:RNA, Transfer, Ser; D011993:Recombinant Fusion Proteins; D010735:Phosphoglycerate Kinase; D001616:beta-Galactosidase