A dual role for zinc fingers in both DNA binding and zinc sensing by the Zap1 transcriptional activator

Abstract

The Zap1 transcriptional activator of Saccharomyces cerevisiae controls zinc homeostasis. Zap1 induces target gene expression in zinc-limited cells and is repressed by high zinc. One such target gene is ZAP1 itself. In this report, we examine how zinc regulates Zap1 function. First, we show that transcriptional autoregulation of Zap1 is a minor component of zinc responsiveness; most regulation of Zap1 activity occurs post-translationally. Secondly, nuclear localization of Zap1 does not change in response to zinc, suggesting that zinc regulates DNA binding and/or activation domain function. To understand how Zap1 responds to zinc, we performed a functional dissection of the protein. Zap1 contains two activation domains. DNA-binding activity is conferred by ®ve C-terminal C 2 H 2 zinc ®ngers and each ®nger is required for highaf®nity DNA binding. The zinc-responsive domain of Zap1 also maps to the C-terminal zinc ®ngers. Furthermore, mutations that disrupt some of these ®ngers cause constitutive activity of a bifunctional Gal4 DNA-binding domain±Zap1 fusion protein.These results demonstrate a novel function of Zap1 zinc ®ngers in zinc sensing as well as DNA binding.

MNR2 Regulates Intracellular Magnesium Storage in Saccharomyces cerevisiae

“…YCpMNR2 was constructed by amplifying a 4.2-kb fragment of the MNR2 gene (including the promoter and terminator regions) from genomic DNA of DY1457 and inserting this fragment into pFL38 via gap repair. The YEpGmycMNR2 plasmid was based on pMYC-Zap1 1-880 (Bird et al 2000), which was modified by replacement of the ZAP1 ORF with the amplified MNR2 ORF via gap repair. The resulting construct contains five repeats of the myc epitope sequence fused to the second codon of the Mnr2 ORF.…”

“…The resulting construct contains five repeats of the myc epitope sequence fused to the second codon of the Mnr2 ORF. Expression of myc-Mnr2 from this plasmid is driven by the upstream activating sequence of the GAL1 promoter fused to a minimal CYC1 promoter (Bird et al 2000). To generate YCpmycMNR2, the myc-Mnr2 ORF was amplified from YEpGmycMNR2 and used to repair the gapped YCpMNR2 plasmid, generating a low-copy construct in which the native MNR2 promoter drives expression of myc-tagged Mnr2.…”

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…Zap1 is the TF for a regulon that responds to limiting amounts of the essential but potentially toxic metal ion Zn +2 (Eide 2009). As shown in Figure 6, Zap1 has two ADs embedded in zinc regulatory regions that function independently of one another in response to the level of Zn +2 (Bird et al 2000). Both ADs have been highly conserved in the Hemiascomycetes, indicating that both are important for Zap1 function.…”

“…AD1 is within the zincresponsive domain (ZRD) that is Cys and His rich (Herbig et al 2005). AD2 is within finger 2 but both fingers 1 and 2 are required for zinc responsiveness (Bird et al 2000).…”

Induction of the ZRC1 Metal Tolerance Gene in Zinc-limited Yeast Confers Resistance to Zinc Shock

“…All three genes are targets of the Zap1 transcriptional activator (18), a central player in zinc homeostasis. Zap1 is active in zinc-limited cells and is repressed by high cytoplasmic/nuclear zinc (19). When active, Zap1p binds to an 11-bp sequence, the zinc response element (ZRE), 1 in the promoters of its target genes (20,21).…”