Saturation mutagenesis of the UASNTR element responsible for GLN3-dependent, nitrogen catabolite repression-sensitive transcriptional activation of allantoin pathway genes in yeast cells identified the dodecanucleotide sequence 5'-TTNCTGATAAGG-3' as the minimum required for UAS activity. There was significant flexibility in mutant sequences capable of supporting UAS activity, which correlates well with the high variation in UASNTR homologous sequences reported to be upstream of the DAL and DUR genes. Three of nine UASNTR-like sequences 5' of the DAL5 gene supported high-level transcriptional activation. The others, which contained nonpermissive substitutions, were not active.
We show that mutation at the GLN3 locus results in decreased steady-state levels of DAL7, DUR1,2, CAR], and URA3 mRNAs derived from cultures grown in the presence of inducer. Basal levels of these RNA species, however, were not significantly affected by a gln3 mutation. The GLN3 product appears to affect gene expression in two ways. The pleiotropic requirement of GLN3 for induced gene expression probably derives from the need of the GLN3 product for inducer uptake into the cell and its loss in gln3 mutants. We also demonstrate that transcriptional activation, mediated by the DALS and DAL7 upstream activation sequences, requires a functional GLN3 gene product. This observation identified transcriptional activation as the most likely point of GLN3 participation in the expression of allantoin system genes.
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