Multiple Signals Regulate GALTranscription in Yeast

Abstract

Gal4p activates transcription of the Saccharomyces GAL genes in response to galactose and is phosphorylated during interaction with the RNA polymerase II (Pol II) holoenzyme. One phosphorylation at S699 is necessary for full GAL induction and is mediated by Srb10p/CDK8 of the RNA Pol II holoenzyme mediator subcomplex. Gal4p S699 phosphorylation is necessary for sensitive response to inducer, and its requirement for GAL induction can be abrogated by high concentrations of galactose in strains expressing wild-type GAL2 and GAL3. Gal4p S699 phosphorylation occurs independently of Gal3p and is responsible for the long-term adaptation response observed in gal3 yeast. SRB10 and GAL3 are shown to represent parallel mechanisms for GAL gene induction. These results demonstrate that Gal4p activity is controlled by two independent signals: one that acts through Gal3p-galactose and a second that is mediated by the holoenzyme-associated cyclindependent kinase Srb10p. Since Srb10p is regulated independently of galactose, our results suggest a function for CDK8 in coordinating responses to specific inducers with the environment through the phosphorylation of gene-specific activators.Eukaryotic cells react to their environment by regulating transcription factors bound to promoters of responsive genes (28). Cells grown in culture are typically provided with sufficient essential nutrients and factors to ensure unchecked propagation. However, in their natural environment, cell growth is ordinarily limited by the scarcity of one or more factors or nutrients. In such circumstances, there must be mechanisms to ensure that transcriptional responses to one signal do not surpass what the cell can accommodate with its limited growth potential. This issue has not yet been addressed in eukaryotes. In this report, we demonstrate that the prototypical transactivator Gal4p is regulated by two separate signals represented by the specific inducer galactose and the RNA polymerase II (Pol II) holoenzyme-associated cyclin-dependent kinase Srb10p/ CDK8. These observations suggest a mechanism whereby responses to a specific inducer can be coordinated with the physiological environment.Gal4p regulates expression of the yeast GAL genes in response to galactose. In noninducing conditions, Gal4p is bound to the upstream activating sequences for galactose (UAS G ) but is prevented from activating transcription by the inhibitor Gal80p (32,43). Rapid induction by galactose requires the product of GAL3 (40, 52, 57), which is a regulatory protein with similarity to the galactokinase encoded by GAL1 (3, 9, 50), although Gal3p does not have galactokinase activity (9). Recent experiments demonstrate that Gal3p, when bound to galactose, directly interacts with Gal80p in the presence of ATP (42,50,59,60). Gal3p-galactose is thought to cause induction of the GAL genes by producing a conformational change in the Gal4p-Gal80p complex that allows interaction of the Gal4p activating domains with the general transcription factors (42, 59). The induced conformation may ...

The S. cerevisiae SAGA complex functions in vivo as a coactivator for transcriptional activation by Gal4

“…The Gal4 activation domain has previously been shown to interact with TBP, TFIIB, and Srb4 (Melcher and Johnston 1995;Wu et al 1996;Ansari et al 1998;Koh et al 1998). In addition, Gal4 becomes phosphorylated upon induction and this phosphorylation is important for Gal4 activation (Rohde et al 2000). Therefore, it seems clear that SAGA is not the only factor important in activation, and multiple interactions are probably required for the normal establishment and maintenance of the activated state.…”

A Fungal Family of Transcriptional Regulators: the Zinc Cluster Proteins

“…Phosphorylation at only a single serine residue (Ser699) in the C-terminal activation domain appears to be necessary for activation (228). However, phosphorylation of Ser699 is not absolutely required for Gal4p activity, since a Ser699-Ala Gal4p mutant shows transcriptional activity in cells lacking Gal80p or in the presence of high galactose levels (219). From these observations, Rohde et al (219) suggested a model in which phosphorylation of Gal4p is required for an acute response to galactose.…”

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

“…TTE1929, TTE1928 and TTE1927 are annotated as GalT, GalK and GalE, respectively, TTE1926 as GalR of the ROK (repressor, open reading frame and kinase) family (Titgemeyer et al, 1994), and TTE1925 as a hypothetical protein. On the other hand, the regulator of this putative operon, GalR, is predicted to be not, as usual, divergently transcribed (Ajdic & Ferretti, 1998;Mackie & Wilson, 1972;Rohde et al, 2000), but in the same direction as the other four genes. Since this was the first investigation of the gal operon in clostridia, it was desirable that the theoretical model could be supported by experimental data.…”

“…Several different uptake systems transport galactose into the cytoplasm, while a unique pathway, the Leloir pathway (Frey, 1996), subsequently converts the galactose to glucose 1-phosphate. The Leloir pathway is known to be composed of gene products from either the lac-gal regulon (Vaillancourt et al, 2002;Vaughan et al, 2001) or the gal operon (Ajdic & Ferretti, 1998;Bettenbrock & Alpert, 1998;Mackie & Wilson, 1972;Rohde et al, 2000;Weickert & Adhya, 1993). The lac-gal regulon consists of the genes involved in lactose and galactose metabolism, such as galRKTEM-lacSZ in Streptococcus thermophilus (Vaughan et al, 2001), while the gal operon contains the genes that participate in galactose metabolism, such as galETKM in Escherichia coli.…”