Isolation of a metal-activated transcription factor gene from Candida glabrata by complementation in Saccharomyces cerevisiae
Zhou, P. | Thiele, D.J.
Metal-inducible transcription of metallothionein (MT) genes involves the interaction of metal-responsive trans-acting factors with specific promoter DNA sequence elements. In this report, we present a genetic selection using the baker's yeast, Saccharomyces cerevisiae, to clone a gene from Candida glabrata encoding a metal-activated DNA-binding protein denoted AMT1. This selection is based on the ability of the AMT1 gene product to activate expression of the C. glabrata MT-1 gene in a copper-sensitive S. cerevisiae host strain. DNA-binding studies using AMT1 protein expressed in Escherichia coli demonstrate that AMT1 is activated by copper or silver to bind to both the MT-I and MT-II promoters of C. glabrata. Sequence comparison of AMT1 protein to the S. cerevisiae copper- or silver-activated DNA-binding protein, ACE1, indicates that AMT1 contains the 11 amino terminal cysteine residues known to be critical for the metal-activated DNA-binding activity of ACE1. In contrast, the carboxyl-terminal portion of AMT1 bears only slight similarity at the primary structure level to the same region of ACE1 known to be important for transcriptional activation. These results suggest that the amino-terminal cysteines, and other conserved residues, play an important role in the ability of AMT1 and ACE1 to sense intracellular copper levels and assume a metal-activated DNA-binding structure.
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