Mapping of the DNA binding domain of the copper-responsive transcription factor Mac1 from Saccharomyces cerevisiae

Mad from Saccharomyces cerevisiae activates transcription of genes, including CTR1 in copper-deficient cells. N-terminal fusions of Mad with the herpes simplex VP16 activation domain were used to show that residues 1-159 in Mac1 constitute the minimal DNA binding domain. Mac1-(1-159) purified from Escherichia coli contains two bound Zn(II) ions. Electrophoretic mobility shift assays showed direct and specific binding by Mac1-(1-159) to a DNA duplex containing the copper-responsive element TTTGCTCA, The DNA binding affinity of Mac1-(1-159) for a duplex containing a single promoter element or an inverted repeat was 5 nM for the 1:1 complex. The N-terminal 40-residue segment of Mad is homologous to the DNA binding zinc module found in the copper-activated transcription factors Ace1 and Amt1. A MAC1 mutation yielding a Cys(11) --> Tyr substitution at the first candidate zinc ligand position relative to Ace1 resulted in a loss of in vivo function, Two TTTGCTCA promoter elements are necessary for efficient Mad-mediated transcriptional activation. The elements appear to function synergistically. Increasing the number of elements yields more than additive enhancements in CTR1 expression.