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

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 pres...

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Bibliographic Details
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
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M000664200