ABSTRACT
In the yeast Saccharomyces cerevisiae, growth with a non-fermentable carbon source requires co-ordinate transcriptional activation of gluconeogenic structural genes by an upstream activation site (UAS) element, designated CSRE (carbon source-responsive element). The zinc cluster protein encoded by CAT8 is necessary for transcriptional derepression mediated by a CSRE. Expression of CAT8 as well as transcriptional activation by Cat8p is regulated by the carbon source, requiring a functional Cat1p (= Snf1p) protein kinase. The importance of both regulatory levels was investigated by construction of CAT8 variants with a constitutive transcriptional activation domain (INO2TAD) and/or a carbon source-independent promoter (MET25 ). Whereas a reporter gene driven by a CSRE-dependent synthetic minimal promoter showed a 40-fold derepression with wild-type CAT8, an almost constitutive expression was found with a MET25-CAT8-INO2TAD fusion construct due to a dramatically increased gene activation under conditions of glucose repression. Similar results were obtained with the mRNA of the isocitrate lyase gene ICL1 and at the level of ICL enzyme activity. Taking advantage of a Cat8p size variant, we demonstrate its binding to the CSRE. Our data show that carbon source-dependent transcriptional activation by Cat8p is the most important mechanism affecting the regulated expression of gluconeogenic structural genes.
Subject(s)
Fungal Proteins/genetics , Fungal Proteins/metabolism , Genes, Fungal , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Trans-Activators/genetics , Trans-Activators/metabolism , Carbon/metabolism , Cell Division/genetics , Gene Expression Regulation, Fungal , Genes, Reporter , Genetic Variation , Glucose/metabolism , Isocitrate Lyase/genetics , Isocitrate Lyase/metabolism , Mutation , Response Elements/genetics , Saccharomyces cerevisiae/metabolism , Transcriptional ActivationABSTRACT
A high-resolution FT infrared spectrum of H3SiF (resolution 0.0024 cm-1) in the region 620-1130 cm-1 was measured and used to analyze the fundamental bands nu2 (A1), 990.851 cm-1; nu3 (A1), 875.011 cm-1; nu5 (E), 962.213 cm-1; and nu6 (E), 729.528 cm-1. A total number of 7241 transition wavenumbers (including 53 perturbation-allowed transitions) with J' = 49 have been fitted by taking into account various Coriolis interactions, alpha-resonance terms, and l-type interactions between and within the vibrational levels v2 = 1, v3 = 1, v5 = 1, and v6 = 1. The strongest interaction in this system of levels is the x-y Coriolis coupling between the v2 = 1 and v5 = 1 vibrational states. However, it turns out that the x-y and z-type Coriolis interactions also have to be introduced explicitly between the v5 = 1 and v6 = 1 states as well as the x-y Coriolis interactions between v3 = 1 and v5 = 1, and v3 = 1 and v6 = 1 states, in order to fit the data quantitatively. The standard deviation of the fit was varsigma = 8.6 x 10(-5) cm-1 for 7241 transition wavenumbers and 68 fitted parameters. The sign relations between the fitted parameters and the possibility of fitting the parameters in different schemes are discussed. The results have been used to find close coincidences between the frequencies of the CO2 laser lines and the transition wavenumbers of the nu2 and nu5 bands of H3SiF for the main natural isotopomer. We report integrated absorption band strengths which are important for infrared laser chemistry. Copyright 1999 Academic Press.
ABSTRACT
The CSRE (carbon source-responsive element) is a sequence motif responsible for the transcriptional activation of gluconeogenic structural genes in Saccharomyces cerevisiae. We have isolated a regulatory gene, DIL1 (derepression of isocitrate lyase, = CAT8), which is specifically required for derepression of CSRE-dependent genes. Expression of CAT8 is carbon source regulated and requires a functional Cat1p (Snf1p) protein kinase. The derepression defect of CAT8 in a cat1 mutant could be suppressed by a mutant Mig1p repressor protein. Derepression of CAT8 also requires a functional HAP2 gene, suggesting a regulatory connection between respiratory and gluconeogenic genes. Carbon source-dependent protein-CSRE complexes detected in a gel retardation analysis with wild-type extracts were absent in cat8 mutant extracts. However, similar experiments with an epitope-tagged CAT8 gene product in the presence of tag-specific antibodies gave evidence against a direct binding of Cat8p to the CSRE. A constitutively expressed GAL4-CAT8 fusion gene revealed a carbon source-dependent transcriptional activation of a UAS(GAL)-containing reporter gene. Activation mediated by Cat8p was no longer detectable in a cat1 mutant. Thus, biosynthetic control of CAT8 as well as transcriptional activation by Cat8p requires a functional Cat1p protein kinase. A model proposing CAT8 as a specific activator of a transcription factor(s) binding to the CSRE is discussed.