An <i>MSC2</i> Promoter-<i>lacZ</i> Fusion Gene Reveals Zinc-Responsive Changes in Sites of Transcription Initiation That Occur across the Yeast Genome

<div><p>The Msc2 and Zrg17 proteins of <i>Saccharomyces cerevisiae</i> form a complex to transport zinc into the endoplasmic reticulum. <i>ZRG17</i> is transcriptionally induced in zinc-limited cells by the Zap1 transcription factor. In this report, we show that <i>MSC2</i> mRNA also increases (~1.5 fold) in zinc-limited cells. The <i>MSC2</i> gene has two in-frame ATG codons at its 5’ end, ATG1 and ATG2; ATG2 is the predicted initiation codon. When the <i>MSC2</i> promoter was fused at ATG2 to the <i>lacZ</i> gene, we found that unlike the chromosomal gene this reporter showed a 4-fold decrease in <i>lacZ</i> 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 <i>MSC2</i>^<i>ATG2</i><i>-lacZ</i> 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, <i>MSC2</i>^<i>ATG2</i><i>-lacZ</i> 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 <i>MSC2</i> 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 <i>MSC2</i>^<i>ATG2</i><i>-lacZ</i> 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.</p>