Translational readthrough at nonsense mutations in the HSF1 gene of Saccharomyces cerevisiae.
The HSF1 gene of Saccharomyces cerevisiae directs the synthesis of the heat shock transcription factor, HSF. The gene is essential; disruption mutations are lethal. Using a plasmid shuffle screen, we isolated mutations in the HSF1 gene after in vitro mutagenesis of plasmid DNA with hydroxylamine. From a collection of both conditional (temperature-sensitive) and unconditional lethal mutations, we recovered mutations that map exclusively to the 5' half of the gene. All are nonsense mutations, including conditional mutations that map 5' to the portion of the HSF1 gene that encodes the DNA-binding domain of the transcription factor. For one such mutation, we demonstrated that the nonsense mutation is subject to translational readthrough, even though there are no known nonsense suppressors in the genetic background of our strain. Our results suggest that the HSF protein is highly tolerant of amino acid changes, a conclusion that is consistent with the very low degree of evolutionary conservation among HSF proteins. Our results also suggest that translational readthrough occurs with moderate efficiency in yeast, particularly when the terminator codon is followed immediately by an A or C residue. This result illustrates that the inference of gene function from mutant phenotype depends critically upon the analysis of a true null allele, and not merely an amber or ochre allele.
Kopczynski
J B
JB
Department of Biology, Indiana University, Bloomington 47405.
Raff
A C
AC
Bonner
J J
JJ
D000595:Amino Acid Sequence; D003062:Codon; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D016153:Genes, Suppressor; D006360:Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017354:Point Mutation; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D003062:Codon; D005656:Fungal Proteins; D006360:Heat-Shock Proteins