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

Abstract

The transcriptional activation response relies on a repertoire of transcriptional activators, which decipher regulatory information through their specific binding to cognate sequences, and their capacity to selectively recruit the components that constitute a given transcriptional complex. We have addressed the possibility of achieving novel transcriptional responses by the construction of a new transcriptional regulator -the Hap2-3-5-Gln3 hybrid modulator -harbouring the HAP complex polypeptides that constitute the DNA-binding domain (Hap2-3-5) and the Gln3 activation domain, which usually act in an uncombined fashion. The results presented in this paper show that transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions is achieved through the action of the novel Hap2-3-5-Gln3 transcriptional regulator. We propose that the combination of the Hap DNA-binding and Gln3 activation domains results in a hybrid modulator that elicits a novel transcriptional response not evoked when these modulators act independently. INTRODUCTIONThe response of Saccharomyces cerevisiae to nutrient limitation is a model whose study has amply contributed to our knowledge of the mechanisms determining transcriptional activation (Zaman et al., 2008). Nutrient limitation results in a complex adaptation of the physiology of yeast cells, which allows them to redefine their transcription programme. This leads to the activation of particular sets of genes, whose products are needed to evoke an appropriate physiological response. Transcriptional activators are composed of a DNA-binding domain, which targets these proteins to specialized binding sites, and an activation domain that mediates transcription initiation; these two domains can be contained in single or different polypeptides (Zaman et al., 2008).When yeast cells are provided with poor nitrogen sources, such as proline, genes coding for enzymes involved in the catabolism of these compounds are highly expressed. 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). Dissociation o...

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