MAPPING OF THE DNA-BINDING DOMAIN OF THE COPPER-RESPONSIVE TRANSCRIPTION FACTOR MAC1 FROM SACCHAROMYCES-CEREVISIAE

Mad from Saccharomyces cerevisiae activates transcription of genes, in cluding CTR1 in copper-deficient cells. N-terminal fusions of Mad with the herpes simplex VP16 activation domain were used to show that resi dues 1-159 in Mac1 constitute the minimal DNA binding domain. Mac1-(1- 159) purified from Escherichia coil contains two bound Zn(II) ions. El ectrophoretic mobility shift assays showed direct and specific binding by Mac1-(1-159) to a DNA duplex containing the copper-responsive elem ent TTTGCTCA, The DNA binding affinity of Mac1-(1-159) for a duplex co ntaining 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 homologou s to the DNA binding zinc module found in the copper-activated transcr iption factors Ace1 and Amt1. A MAC1 mutation yielding a Cys(11) --> T yr substitution at the first candidate zinc ligand position relative t o Ace1 resulted in a loss of in vivo function, Two TTTGCTCA promoter e lements are necessary for efficient Mad-mediated transcriptional activ ation. The elements appear to function synergistically. Increasing the number of elements yields more than additive enhancements in CTR1 exp ression.