Transcription Initiation of the Yeast IMD2 Gene Is Abolished in Response to Nutrient Limitation through a Sequence in Its Coding Region

Abstract

The yeast IMD2 to IMD4 and GUA1 genes, involved in GMP synthesis, are highly expressed in exponentially growing cells but are shut off when cells cease to grow upon nutrient limitation. We show for the IMD2 gene that this effect is not specific to certain carbon sources or to growth rate. Strikingly, the cis elements responsible for this nutritional response are contained within a 23-nucleotide sequence in the coding region of the IMD2 gene. Despite its very unusual location, this regulatory sequence mediates the repression of transcription initiation. From our data, we conclude that GMP synthesis is downregulated upon nutrient limitation through an active mechanism. We show that this transcriptional shutoff abolishes any possibility of the induction of IMD2, even under drastic conditions of guanylic nucleotide limitation. Taken together, these results indicate that low levels of guanylic nucleotides could be required for proper entry into stationary phase.GMP synthesis from IMP results from two successive enzymatic reactions catalyzed by IMP dehydrogenase (IMPDH) and GMP synthetase (Fig. 1A). This metabolic pathway is highly conserved through evolution; sequencing of the Saccharomyces cerevisiae genome has revealed four homologs of IMPDH-encoding genes (IMD1 to IMD4), while the second step of the pathway requires the product of the GUA1 gene (6). Guanylic nucleotide level feedback regulates the transcription of the IMD genes (7). Indeed, the addition of guanine, a precursor of guanylic nucleotides, represses IMD2 transcription (7), while the addition of inhibitors of GMP synthesis, such as mycophenolic acid (MPA) or 6-azauracil, strongly induces IMD2 transcription (7,25). A specific sequence, called the guanine response element (GRE), was identified in the promoter of IMD2 and found to be required for this regulation (7). Besides this specific regulation by the end product(s) of the pathway, IMD2 expression was also found to respond to growth phase. Indeed, microarray experiments revealed that the expression of IMD2 decreases when cells enter stationary phase (5, 9), and this result was confirmed by Northern blot analysis (26); however, the mechanism leading to this regulation has not been yet elucidated.While the transcription of many genes decreases during stationary phase, understanding of this phenomenon is still incomplete. It is important to find out whether this regulation is the "passive" result of a general decrease in transcriptional efficiency or whether it indeed is an "active" regulation mechanism. It is clear that transcription is not simply abolished during stationary phase, since a subset of genes is induced during this phase (5, 9). Interestingly, mutations affecting the induction of YGP1, a gene induced by nutrient deprivation and entry into stationary phase, also affect the expression of ACT1, a gene repressed in stationary phase (1). Therefore, common mechanisms could regulate increased or decreased expression of specific genes in stationary phase in response to metabolic signals.Because dec...

The Critical cis-Acting Element Required for IMD2 Feedback Regulation by GDP Is a TATA Box Located 202 Nucleotides Upstream of the Transcription Start Site

“…Plasmid P892 or P913, containing the IMD2-lacZ fusions and 311 bp upstream of the IMD2 ATG, corresponding to 205 bp of the IMD2 promoter, and either a wild-type (TATAAATA) or a mutated (TACCCATA) GRE box, respectively, was described elsewhere (10). The IMD2-lacZ plasmid P855 or P1200, with 355 bp upstream of the IMD2 ATG and either 1,135 bp or only the ATG of the coding region, respectively, was described elsewhere (10,12). The lacZ fusions were constructed in the URA3 vectors described by Myers and coworkers (25).…”

“…It has been demonstrated that the levels of IMD transcripts decrease when cell growth ceases in response to nutrient limitation (8,12,13,30). Additionally, in exponentially growing cells, IMD transcripts are feedback regulated by pathway end products (10,29).…”

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…A mutagenesis of the IMD2 gene revealed that the sequence involved in growth phase regulation lies in the coding region of the gene (Escobar-Henriques et al 2003a). Somehow this sequence affects recruitment of the TATA-box-binding protein to the upstream IMD2 promoter Kurtz et al (1999) regions (Escobar-Henriques et al 2003a), but the precise mechanisms have not yet been elucidated. For adenine deaminase (Aah1), regulation upon entry into stationary phase occurs both at the transcriptional and the post-transcriptional levels.…”

“…In yeast, expression of IMPDH-coding genes (IMDs) is strongly affected by growth phase (Shaw et al 2001;Escobar-Henriques et al 2003a). A mutagenesis of the IMD2 gene revealed that the sequence involved in growth phase regulation lies in the coding region of the gene (Escobar-Henriques et al 2003a). Somehow this sequence affects recruitment of the TATA-box-binding protein to the upstream IMD2 promoter Kurtz et al (1999) regions (Escobar-Henriques et al 2003a), but the precise mechanisms have not yet been elucidated.…”

“…Interestingly, in Bacillus subtilis, diminution of intracellular GTP concentration is required for proper entry into stationary phase (Ratnayake-Lecamwasam et al 2001). In yeast, expression of IMPDH-coding genes (IMDs) is strongly affected by growth phase (Shaw et al 2001;Escobar-Henriques et al 2003a). A mutagenesis of the IMD2 gene revealed that the sequence involved in growth phase regulation lies in the coding region of the gene (Escobar-Henriques et al 2003a).…”