Mapping the DNA binding domain of the Zap1 zinc-responsive transcriptionalactivator

The Zap1 transcriptional activator of Saccharomyces cerevisiae plays a majo r role in zinc homeostasis by inducing the expression of several genes unde r zinc-limited growth conditions. This activation of gene expression is med iated by binding of the protein to one or more zinc-responsive elements pre sent in the promoters of its target genes. To better understand how Zap1 fu nctions, we mapped its DNA binding domain using a combined in vivo and in v itro approach. Our results show that the Zap1 DNA binding domain maps to th e carboxyl-terminal 194 amino acids of the protein; this region contains fi ve of its seven potential zinc finger domains. Fusing this region to the Ga 14 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-direct ed mutagenesis, we demonstrated that each of the five carboxyl-terminal zin c fingers are required for high affinity DNA binding.