Abstract
Zinc homeostasis in yeast is controlled primarily through the regulation of zinc uptake. Transcription of the ZRT1 and ZRT2 zinc transporters increases in zinc-limited cells, and this induction is dependent on the ZAP1 gene. We hypothesized previously that ZAP1 encodes a zinc-responsive transcriptional activator. Expression of ZAP1 itself increases in zinc-limited cells. This response is also dependent on ZAP1 function through a potential positive autoregulatory mechanism. In this report, we describe the characterization of zinc-responsive elements (ZREs) in the promoters of the ZRT1, ZRT2, and ZAP1 genes. A ZRE consensus sequence, 5'-ACCYYNAAGGT-3', was identified and found to be both necessary and sufficient for zinc-responsive transcriptional regulation. We also demonstrate that ZREs are DNA binding sites for ZAP1. First, a dominant ZAP1 mutation, ZAP1-1(up), which causes increased expression of ZAP1-regulated genes in zinc-replete cells, exerted its effects specifically through the ZREs. Second, electrophoretic mobility shift assays and in vitro DNase I footprint analyses indicated that ZAP1 binds to ZREs in a sequence-specific fashion. These studies demonstrate that ZAP1 plays a direct role in controlling zinc-responsive gene expression in yeast by binding to zinc-responsive elements in the promoters of genes that it regulates.
Highlights
The variety of roles that zinc plays in cellular processes is a prime example of the utility of metal ions in biology
We demonstrate that ZAP1 is the transcriptional activator that directly controls zinc-responsive gene expression in yeast and identify its binding sites in the promoters of the ZRT1, ZRT2, and ZAP1 genes
The ZAP1 gene is required for zinc-responsive gene expression in yeast [10]
Summary
The variety of roles that zinc plays in cellular processes is a prime example of the utility of metal ions in biology. The second system is encoded by the ZRT2 gene and has lower affinity for zinc [8] Both ZRT1 and ZRT2 are regulated by zinc availability; zinc limitation induces ZRT1 and ZRT2 transcription, whereas growth under zinc-replete conditions represses their expression [9, 10]. This zinc-responsive transcriptional regulation requires the activity of the ZAP1 gene [10]. From our initial characterization of ZAP1, we proposed that this gene encodes a transcriptional activator, the function of which is repressed by zinc. This hypothesis was based on several observations. We found that ZAP1 was required to increase its own transcription in response to zinc, potentially through a positive autoregulatory mechanism
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