Jensen L T, Posewitz M C, Srinivasan C, Winge D R
Departments of Medicine and Biochemistry, University of Utah Health Science Center, Salt Lake City, Utah 84132, USA.
J Biol Chem. 1998 Sep 11;273(37):23805-11. doi: 10.1074/jbc.273.37.23805.
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.
来自酿酒酵母的Mac1可激活包括铜缺乏细胞中的CTR1在内的基因转录。将Mac1与单纯疱疹病毒VP16激活结构域进行N端融合，以表明Mac1中的1 - 159位残基构成最小DNA结合结构域。从大肠杆菌中纯化的Mac1-(1 - 159)含有两个结合的Zn(II)离子。电泳迁移率变动分析表明Mac1-(1 - 159)与含有铜反应元件TTTGCTCA的DNA双链体直接且特异性结合。Mac1-(1 - 159)对含有单个启动子元件或反向重复序列的双链体的DNA结合亲和力，对于1:1复合物为5 nM。Mac1的N端40个残基片段与铜激活转录因子Ace1和Amt1中发现的DNA结合锌模块同源。相对于Ace1，在第一个候选锌配体位置产生Cys11→Tyr取代的MAC1突变导致体内功能丧失。两个TTTGCTCA启动子元件对于有效的Mac1介导的转录激活是必需的。这些元件似乎协同发挥作用。增加元件数量会使CTR1表达产生超过累加效应的增强。
