Mapping the DNA Binding Domain of the Zap1 Zinc-responsive Transcriptional Activator

• Published in: The Journal of biological chemistry 2000-05, Vol.275 (21), p.16160-16166
• Main Authors: Bird, Amanda, Evans-Galea, Marguerite V., Blankman, Elizabeth, Zhao, Hui, Luo, Huan, Winge, Dennis R., Eide, David J.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites - genetics; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fungal Proteins - genetics; Gene Expression; Genes, Reporter; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments - metabolism; Recombinant Fusion Proteins - genetics; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Trans-Activators - chemistry; Trans-Activators - genetics; Transcription Factors - genetics; Transcriptional Activation; Zap1 protein; Zinc - metabolism; Zinc Fingers - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M000664200

The Zap1 transcriptional activator ofSaccharomyces 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 zap1Δ 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.