Mapping the DNA binding domain of the Zap1 zinc-responsive transcriptional activator.
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.
Bird
A
A
Department of Nutritional Sciences, University of Missouri, Columbia, Missouri 65211, USA.
Evans-Galea
M V
MV
Blankman
E
E
Zhao
H
H
Luo
H
H
Winge
D R
DR
Eide
D J
DJ
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C103264:ZAP1 protein, S cerevisiae; D015032:Zinc