Synergistic operation of four -acting elements mediate high level transcription in

The Saccharomyces cerevisiae allantoate/ureidosuccinate permease gene (DAL5) is often used as a reporter in studies of the Tor1/2 protein kinases which are specifically inhibited by the clinically important immunosuppressant and anti-neoplastic drug, rapamycin. To date, only a single type of cis-acting element has been shown to be required for DAL5 expression, two copies of the GATAA-containing UASNTR element that mediates nitrogen catabolite repression-sensitive transcription. UASNTR is the binding site for the transcriptional activator, Gln3 whose intracellular localization responds to the nitrogen supply, accumulating in the nuclei of cells provided with poor nitrogen sources and in the cytoplasm when excess nitrogen is available. Recent data raised the possibility that DAL5 might also be regulated by the retrograde system responsible for control of early TCA cycle gene expression, prompting us to investigate the structure of the DAL5 promoter in more detail. Here, we show that clearly one (UASB), and possibly two (UASA), additional cis-acting elements are required for full DAL5 expression. One of these elements (UASB) is in a region that is heavily protected from DNaseI digestion and functions in a highly synergistic manner with the two UASNTR elements. Cis-acting elements UASNTR–UASA and UASNTR–UASB are situated on the same face of the DNA two and one turn apart, respectively. We also found that decreased DAL5 expression in glutamate-grown cells, a characteristic shared with retrograde regulation, likely derives from decreased nuclear Gln3 levels that occur under these growth conditions rather than direct retrograde system control.