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

Citation
A. Bird et al., Mapping the DNA binding domain of the Zap1 zinc-responsive transcriptionalactivator, J BIOL CHEM, 275(21), 2000, pp. 16160-16166
Citations number
19
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
0021-9258 → ACNP
Volume
275
Issue
21
Year of publication
2000
Pages
16160 - 16166
Database
ISI
SICI code
0021-9258(20000526)275:21<16160:MTDBDO>2.0.ZU;2-Q
Abstract
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.