Recognition of nitrogen-responsive upstream activation sequences of Saccharomyces cerevisiae by the product of the GLN3 gene

Abstract

We describe the purification of the product of the GLN3 gene of Saccharomyces cerevisiae and the demonstration that the purified product, Gln3p, binds specifically to the DNA sequences GATAAG and GATTAG, previously identified as nitrogen-responsive upstream activation sequences (UAS N ). When Gln3p is overproduced, it is released from the cells in a highly aggregated form incapable of specific binding to UAS N . We used Gln3p tagged with six histidine codons at the 5 terminus and equipped with a galactose-inducible promoter to overproduce histidine-tagged Gln3p. The material was denatured, adsorbed to an Ni-nitrilotriacetic acid (NTA)-agarose column, eluted with imidazole, and after renaturation further purified on a gel filtration column. We then demonstrated the specific binding of the more than 90% pure Gln3p to the UAS N by gel shift and footprinting methods.

Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine

“…Why did Gln3 constitutively localize to the nucleus when residues 336-345 were abolished but did not behave similarly in the three substitution mutants we constructed, in particular, 332dddd Gln3-Myc 13 (pRR787)? To address this question, we looked in detail at the amino acid sequence around Gln3 residues 336-345 and discovered that they were immediately adjacent to the Gln3 zinc finger motif responsible for Gln3 binding to its target GATA elements in NCR-sensitive gene promoters Rai et al 1989;Bysani et al 1991;Blinder and Magasanik 1995;Cunningham et al 1996;Stanbrough and Magasanik 1996). This entire region of Gln3, residues 306-357, is highly conserved in both closely and more distantly related yeasts ( Figure 8A).…”

Ammonia Regulates VID30 Expression and Vid30p Function Shifts Nitrogen Metabolism toward Glutamate Formation Especially when Saccharomyces cerevisiae Is Grown in Low Concentrations of Ammonia

“…1-4 for reviews of the field). When nitrogen availability limits growth, as occurs with poor nitrogen sources such as proline, Gln3p and/or Gat1p bind to the GATA sequences upstream of NCR-sensitive genes and activate their transcription (5)(6)(7)(8)(9)(10)(11). However, in nitrogen excess, the GATA sites are unoccupied because Gln3p and Gat1p are excluded from the nucleus (12)(13)(14)(15).…”

Hap2-3-5-Gln3 determine transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions in the yeast Saccharomyces cerevisiae

“…Conversely, in the presence of high-quality nitrogen sources such as glutamine, a decrease in the levels of catabolic enzymes is observed. The reduced expression of the genes coding for enzymes involved in the utilization of poor nitrogen sources is brought about through the action of a regulatory system known as nitrogen catabolite repression (NCR) (Blinder & Magasanik, 1995;Coffman et al, 1995Coffman et al, , 1996Courchesne & Magasanik, 1988;Minehart & Magasanik, 1991) NCR operates through the action of two transcriptional activators, Gln3 and Nil1/ Gat1, and two repressors, Dal80 and Gzf3/Deh1/Nil2 (Minehart & Magasanik, 1991;Stanbrough et al, 1995;Svetlov & Cooper, 1998;Magasanik & Kaiser, 2002), whose expression is regulated by nitrogen levels. In the presence of repressive nitrogen sources, the TOR signalling pathway promotes the association of the GATA transcription factors Gln3 and Gat1 with the cytoplasmic protein Ure2, thus retaining Gln3 and Gat1 in the cytoplasm (Beck & Hall, 1999;Cardenas et al, 1999;Hardwick et al, 1999).…”