Mapping of the DNA binding domain of the copper-responsive transcription factor Mac1 from Saccharomyces cerevisiae.
Mac1 from Saccharomyces cerevisiae activates transcription of genes, including CTR1 in copper-deficient cells. N-terminal fusions of Mac1 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 Mac1 is homologous to the DNA binding zinc module found in the copper-activated transcription factors Ace1 and Amt1. A MAC1 mutation yielding a Cys11 --> 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 Mac1-mediated transcriptional activation. The elements appear to function synergistically. Increasing the number of elements yields more than additive enhancements in CTR1 expression.
Jensen
L T
LT
Departments of Medicine and Biochemistry, University of Utah Health Science Center, Salt Lake City, Utah 84132, USA.
Posewitz
M C
MC
Srinivasan
C
C
Winge
D R
DR
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism*; D003545:Cysteine; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine; D015032:Zinc / Q000378:metabolism
D005656:Fungal Proteins; C084954:MAC1 protein, S cerevisiae; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014443:Tyrosine; D003300:Copper; D015032:Zinc; D003545:Cysteine