RESUMO
The carbon source-responsive element (CSRE) functions as an activating promoter motif of gluconeogenic genes in Saccharomyces cerevisiae. The positively acting regulatory genes CAT8 and SIP4 encode CSRE-binding proteins which contribute unequally to the regulated expression of a CSRE-dependent reporter gene (85% and 15%, respectively, under conditions of glucose derepression). Deregulated variants of Cat8 and Sip4 are able to bind to the CSRE and allow glucose-insensitive gene activation, even in the absence of the other protein, arguing against the physiological significance of heterodimer formation. Gel retardation assays provide evidence for a different binding affinity of Cat8 and Sip4 to at least some CSRE sequence variants. Both efficient biosynthesis of and transcriptional activation by Sip4 require a functional CAT8 gene, while Cat8 was not dependent on SIP4. Thus, our data suggest that the apparent minor importance of Sip4 may be the result of autoregulatory cross-talk among the isofunctional activators Cat8 and Sip4. The derepression deficiency of a CSRE-dependent reporter gene in a strain lacking the Cat1 (Snf1) protein kinase can be suppressed by Sip4 fused to a strong heterologous activation domain. This finding agrees with the idea that phosphorylation by Cat1 may convert Sip4 into a functional activator.
Assuntos
Carbono/metabolismo , Gluconeogênese/genética , Elementos de Resposta/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transativadores/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dosagem de Genes , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Genes Reporter , Variação Genética , Genótipo , Mutação , Fenótipo , Plasmídeos/genética , Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Transcrição Gênica/genética , Ativação TranscricionalRESUMO
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.
Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transativadores/genética , Transativadores/metabolismo , Carbono/metabolismo , Divisão Celular/genética , Regulação Fúngica da Expressão Gênica , Genes Reporter , Variação Genética , Glucose/metabolismo , Isocitrato Liase/genética , Isocitrato Liase/metabolismo , Mutação , Elementos de Resposta/genética , Saccharomyces cerevisiae/metabolismo , Ativação TranscricionalRESUMO
The yeast Saccharomyces cerevisiae contains two acetyl-CoA synthetase genes, ACS1 and ACS2. While ACS1 transcription is glucose repressible, ACS2 shows coregulation with structural genes of fatty acid biosynthesis. The ACS2 upstream region contains an ICRE (inositol/choline-responsive element) as an activating sequence and requires the regulatory genes INO2 and INO4 for maximal expression. We demonstrate in vitro binding of the heterodimeric activator protein Ino2p/Ino4p to the ACS2 promoter. In addition, the pleiotropic transcription factor Abf1p also binds to the ACS2 control region. The identification of ACS2 activating elements also found upstream of ACC1, FAS1 and FAS2 suggests a role of this acetyl-CoA synthetase isoenzyme for the generation of the acetyl-CoA pool required for fatty acid biosynthesis.
