Two of the five zinc fingers in the Zap1 transcription factor DNA binding domain dominate site-specific DNA binding
Two of the five zinc fingers in the Zap1 transcription factor DNA binding domain dominate site-specific DNA binding
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DOI：
10.1021/bi0263199
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发表时间：
2003-02-04
期刊：
影响因子：
2.9
通讯作者：
Winge, DR
中科院分区：
文献类型：
--
作者：
Evans-Galea, MV;Blankman, E;Winge, DR
来自酿酒酵母的Zap 1转录激活因子在锌缺乏条件下诱导一系列含有11个碱基对的保守启动子元件（ZRE）的基因的表达。这项工作表明，Zap 1使用其七个锌指结构域中的四个与ZRE接触，其中两个通过与必需的ACC-GGT末端接触来主导相互作用。两个锌指结构域（ZF 1和ZF 2）不接触DNA，第三个ZF 3可能对指间蛋白质-蛋白质相互作用更重要。从Zap 1的三重突变中鉴定出对ZRE接触重要的锌指结构域，改变每个指中α螺旋中已知对锌指蛋白中的DNA接触重要的三个残基。ZF 4和ZF 7中-1，3和6螺旋残基的替换降低了Zap 1对野生型ZRE的亲和力。相比之下，在间插的ZF 5和ZF 6结构域内的三重突变具有最小的影响。数据表明，指4和7接触ACC-GGT末端，而指5和6接触5 bp中心ZRE序列。这一结论是证实了Zap I的亲和力降低ZRE DNA双链体含有突变的AC-GT末端的ZRE，而颠换突变内的中央5 bp的ZRE Zap 1结合亲和力的影响最小。
The Zap1 transcriptional activator from Saccharomyces cerevisiae induces expression of a series of genes containing an 11 base pair conserved promoter element (ZRE) under conditions of zinc deficiency. This work shows that Zap1 uses four of its seven zinc finger domains to contact the ZRE and that two of these dominate the interaction by contacting the essential ACC-GGT ends. Two Zn finger domains (ZF1 and ZF2) do not contact DNA, and a third ZF3 may be more important for interfinger protein-protein interactions. Zn finger domains important for ZRE contact were identified from triple mutations in Zap1, changing three residues in the alpha helix in each finger known to be important for DNA contacts in Zn finger proteins. Replacement of -1, 3, and 6 helix residues in ZF4 and ZF7 reduced the affinity of Zap1 for the wild-type ZRE. In contrast, triple mutations within the intervening ZF5 and ZF6 domains had minimal effect. The data argue that fingers 4 and 7 contact the ACC-GGT ends while fingers 5 and 6 contact the 5 bp central ZRE sequence. This conclusion is corroborated by decreased Zap I affinity for a ZRE DNA duplex containing mutations of the AC-GT ends of the ZRE, whereas transversion mutations within the central 5 bp of the ZRE had minimal effect on Zap1 binding affinity.