Copper metallothionein of yeast, structure of the gene, and regulation of expression
Key findings
Butt et al. determined the nucleotide sequence of the yeast CUP1 (Cu-MT) gene, identifying a 186-nucleotide open reading frame encoding a 61-amino-acid, 6573-dalton protein. The protein contained 12 cysteine residues comprising 20% of total amino acids, lower than the 32% in vertebrate metallothioneins. Two TATA elements at positions -98 and -138 upstream of the ATG, and an AATAAA polyadenylation signal at +377, defined the transcriptional architecture.
Cu-MT mRNA was induced 50-fold by 0.5 mM CuSO4 in copper-sensitive yeast transformed with the high-copy plasmid YEp36. Induction was copper-specific: zinc, cadmium, and gold at comparable concentrations failed to activate transcription, distinguishing the CUP1 locus from vertebrate metallothionein promoters, which respond to both cadmium and zinc.
A 431-bp BamHI-RsaI fragment upstream of the Cu-MT coding region was fused to the E. coli galK reporter gene in the single-copy plasmid pYSK12. Galactokinase activity increased 50-fold at 100 µM CuSO4, confirming that the 431-bp upstream region was sufficient for copper-inducible heterologous gene expression in yeast. Zinc, cadmium, and gold did not activate galK, establishing the Cu-MT regulatory sequences as a copper-specific, tunable promoter.