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 Gal4 activation domain complemented a zap1Delta 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.

• PMID: 10747942
• DOI full text
• PubMed
• PubTator

Reference Type

Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Bird A, Evans-Galea MV, Blankman E, Zhao H, Luo H, Winge DR, Eide DJ

Primary Lit For

Additional Lit For

Review For