Abstract
The Msc2 and Zrg17 proteins of Saccharomyces cerevisiae form a complex to transport zinc into the endoplasmic reticulum. ZRG17 is transcriptionally induced in zinc-limited cells by the Zap1 transcription factor. In this report, we show that MSC2 mRNA also increases (~1.5 fold) in zinc-limited cells. The MSC2 gene has two in-frame ATG codons at its 5’ end, ATG1 and ATG2; ATG2 is the predicted initiation codon. When the MSC2 promoter was fused at ATG2 to the lacZ gene, we found that unlike the chromosomal gene this reporter showed a 4-fold decrease in lacZ mRNA in zinc-limited cells. Surprisingly, β-galactosidase activity generated by this fusion gene increased ~7 fold during zinc deficiency suggesting the influence of post-transcriptional factors. Transcription of MSC2ATG2-lacZ was found to start upstream of ATG1 in zinc-replete cells. In zinc-limited cells, transcription initiation shifted to sites just upstream of ATG2. From the results of mutational and polysome profile analyses, we propose the following explanation for these effects. In zinc-replete cells, MSC2ATG2-lacZ mRNA with long 5’ UTRs fold into secondary structures that inhibit translation. In zinc-limited cells, transcripts with shorter unstructured 5’ UTRs are generated that are more efficiently translated. Surprisingly, chromosomal MSC2 did not show start site shifts in response to zinc status and only shorter 5’ UTRs were observed. However, the shifts that occur in the MSC2ATG2-lacZ construct led us to identify significant transcription start site changes affecting the expression of ~3% of all genes. Therefore, zinc status can profoundly alter transcription initiation across the yeast genome.
Highlights
Zinc is an essential nutrient for function of the secretory pathway by serving as a cofactor for both secreted proteins and proteins that reside in secretory pathway compartments
The importance of regulatory mechanisms for maintaining zinc homeostasis in the secretory pathway of Saccharomyces cerevisiae was recently demonstrated by studies of Zrg17, a zinc
This was motivated by our interest in an integrated understanding of the regulation of zinc homeostasis in the early secretory pathway
Summary
Zinc is an essential nutrient for function of the secretory pathway by serving as a cofactor for both secreted proteins and proteins that reside in secretory pathway compartments. The importance of regulatory mechanisms for maintaining zinc homeostasis in the secretory pathway of Saccharomyces cerevisiae was recently demonstrated by studies of Zrg, a zinc. Zinc Status Alters Transcription Initiation Sites in Yeast transporter in the endoplasmic reticulum (ER) that supplies zinc to that organelle. ZRG17 gene expression is induced in zinc-limited cells and this gene is a direct target gene of the Zap transcription factor [1]. Zap is the central player in zinc homeostasis where it activates the expression of ~80 genes in response to zinc deficiency [2]. Disruption of Zap1-mediated induction of ZRG17 under zinc-limited conditions led to elevated ER stress, demonstrating the biological importance of this regulation to maintaining ER function
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