Copper metallothionein of yeast, structure of the gene, and regulation of expression

Butt et al. (1984). PNAS DOI: 10.1073/pnas.81.11.3332 Citations: 286

Regulatory DNA controlling copper induction of yeast metallothionein (Cu-MT) was unknown. This paper sequenced the Cu-MT gene and fused its upstream region to the E. coli galK reporter. Copper induced galK expression 50-fold, while zinc, cadmium, and gold did not.

Parts used

CUP1 Promoter

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.

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Abstract

Addition of copper to yeast cells leads to the induction of a low molecular weight, cysteine-rich protein that binds copper. This protein, termed copper chelatin or thionein, is related to the metallothionein family of proteins that are induced in response to cadmium and zinc in vertebrate cells. We have determined the structure of the yeast copper-binding protein by DNA sequence analysis of the gene. Although the 6573-dalton yeast protein is substantially divergent from vertebrate metallothioneins, the arrangement of 12 cysteine residues, which is a hallmark of metal-binding proteins, is partially conserved. We analyzed the regulatory DNA sequence of the gene by fusing it with the Escherichia coli galactokinase gene and assaying the levels of enzyme activity in yeast in response to copper. The transcriptional activation has a specific requirement for copper. Zinc, cadmium, and gold were unable to regulate the galactokinase activity. The yeast copper metallothionein regulatory sequences represent a previously unreported class of yeast promoter that is regulated by copper.