We have isolated the NILI gene, whose product is an activator of the transcription of nitrogen-regulated genes, by virtue of the homology of its zinc-finger domain to that of the previously identified activator, the product of GLN3. Disruption of the chromosomal NILI gene enabled us to compare the effects of Gln3p and of Nillp on the expression of the nitrogen-regulated genes GLN1, GDH2, and GAP), coding respectively for glutamine synthetase, NAD-linked glutamate dehydrogenase, and general amino acid permease. Our results show that the nature of GATAAG sequences that serve as the upstream activation sequence elements for these genes determines their abilities to respond to Gln3p and Nillp. The results further indicate that Gln3p is inactivated by an increase in the intracellular concentration of glutamine and that Nillp is inactivated by an increase in intracellular glutamate.The presence of glutamine in the growth medium of Saccharomyces cerevisiae prevents the expression of GLN] and of GDH2, the structural genes for glutamine synthetase and the NADI-linked glutamate dehydrogenase (1). In a medium containing glutamate as a source of nitrogen, the expression of these genes requires the product of GLN3, Gln3p, a protein containing a zinc finger domain homologous to that of GATA factors of avian and mammalian cells, capable of binding to the sequences 5'-GATAAGATAAG-3' and 5'-GATTAGAT-TAG-3' located upstream of GLN1 and GDH2, respectively (1, 2). Deletion of these sequences greatly reduces the expression of these genes. The sequence 5'-GATAA-3' is also found upstream of other nitrogen-regulated genes and is apparently responsible for the activation of the expression of these genes by Gln3p. Another protein, the product of URE2, disables Gln3p in response to an increase in the intracellular concentration of glutamine. Mutants lacking Ure2p express Gln3p-activated genes in the presence of glutamine (1).Among the genes whose transcription can be activated by Gln3p is GAP], the structural gene for the general amino acid permease. This gene is not expressed in media containing glutamine, and the lack of Gln3p greatly diminished its expression in a medium containing glutamate as the source of nitrogen. Nevertheless, the lack of Gln3p did not prevent strong expression of GAP1 in media containing ammonia or urea as source of nitrogen. Apparently, a transcription factor other than Gln3p, which we have named Nillp, is responsible for the activation of transcription of GAP] in media containing these sources of nitrogen (3). Elimination of Ure2p enabled Gln3p, but not Nillp, to activate the transcription of GAP1 in the presence of glutamine (4).Examination of the DNA sequence revealed the presence of five GATAAG sequences within the 650-bp region preceding the translational start site of GAP1. This region was analyzed by making partial deletions from the 5' and 3' ends, fusing the remainder to CYCl-lacZ, and examining the 3-galactosidase levels in wild-type and gln3 mutant cells carrying plasmids containing these fusi...

The cellular level and activity of the general amino acid permease, the product of the GAP1 gene of Saccharomyces cerevisiae, are regulated at the level of transcription by two systems, the products of URE2/GLN3 and NIL1 in response to the nitrogen sources of the growth medium and inactivation in response to the presence of glutamine or glutamate. Active permease is phosphorylated. The addition of glutamine causes rapid dephosphorylation and inactivation of the permease with the same kinetics, which is followed by slower disappearance of the protein. These results suggest that inactivation of the permease results from its dephosphorylation.

We present an analysis of the DNA region located upstream of GAP1, the structural gene for the general amino acid permease, which contains the sites required for activation of transcription of this gene in response to the nitrogen source of the growth medium. This gene is not expressed in media containing glutamine, and its transcription is activated in response to Gln3p in cells using glutamate as the source of nitrogen and by Nil1p in cells using urea as the source of nitrogen. We show that full response to both activators requires the presence of two GATAAG sites, as well as the presence of auxiliary sites located in the interval between 602 and 453 bp from the translational start site. The fact that both Gln3p and Nil1p utilize GATAAG sites to activate transcription is reflected in the high homology of the zinc finger regions of the two proteins.