Mapping the DNA binding domain of the Zap1 zinc-responsive transcriptional activator
Mapping the DNA binding domain of the Zap1 zinc-responsive transcriptional activator
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DOI：
10.1074/jbc.m000664200
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发表时间：
2000-05-26
影响因子：
4.8
通讯作者：
Eide, DJ
中科院分区：
文献类型：
--
作者：
Bird, A;Evans-Galea, MV;Eide, DJ
酿酒酵母的Zap 1转录激活因子通过在锌限制生长条件下诱导几个基因的表达在锌稳态中起主要作用。这种基因表达的激活通过蛋白质与其靶基因启动子中存在的一个或多个锌响应元件的结合来介导。为了更好地了解Zap 1的功能，我们使用体内和体外相结合的方法绘制了其DNA结合结构域。我们的研究结果表明，Zap 1 DNA结合域映射到羧基端的194个氨基酸的蛋白质，这个区域包含五个潜在的锌指结构域的七个。将该区域融合到Ga 14激活结构域补充了zap 1 Delta突变以实现低锌生长，并且还赋予锌响应元件-lacZ报告基因高水平表达。在体外，纯化的194个残基的片段结合到DNA的高亲和力（解离常数在低纳摩尔范围内）类似于较长的片段Zap 1。此外，通过缺失和定点突变，我们证明了五个羧基末端锌指中的每一个都是高亲和力DNA结合所必需的。
The Zap1 transcriptional activator of Saccharomyces cerevisiae plays a major role in zinc homeostasis by inducing the expression of several genes under zinc-limited growth conditions. This activation of gene expression is mediated by binding of the protein to one or more zinc-responsive elements present in the promoters of its target genes. To better understand how Zap1 functions, we mapped its DNA binding domain using a combined in vivo and in vitro approach. Our results show that the Zap1 DNA binding domain maps to the carboxyl-terminal 194 amino acids of the protein; this region contains five of its seven potential zinc finger domains. Fusing this region to the Ga14 activation domain complemented a zap1 Delta mutation for low zinc growth and also conferred high level expression on a zinc-responsive element-lacZ reporter. In vitro, the purified 194-residue fragment bound to DNA with a high affinity (dissociation constant in the low nanomolar range) similar to that of longer fragments of Zap1. Furthermore, by deletion and site-directed mutagenesis, we demonstrated that each of the five carboxyl-terminal zinc fingers are required for high affinity DNA binding.