RESUMO
Mediator is an evolutionary conserved coregulator complex required for transcription of almost all RNA polymerase II-dependent genes. The Schizosaccharomyces pombe Mediator consists of two dissociable components-a core complex organized into a head and middle domain as well as the Cdk8 regulatory subcomplex. In this work we describe a functional characterization of the S. pombe Mediator. We report the identification of the S. pombe Med20 head subunit and the isolation of ts alleles of the core head subunit encoding med17+. Biochemical analysis of med8(ts), med17(ts), Deltamed18, Deltamed20 and Deltamed27 alleles revealed a stepwise head domain molecular architecture. Phenotypical analysis of Cdk8 and head module alleles including expression profiling classified the Mediator mutant alleles into one of two groups. Cdk8 module mutants flocculate due to overexpression of adhesive cell-surface proteins. Head domain-associated mutants display a hyphal growth phenotype due to defective expression of factors required for cell separation regulated by transcription factor Ace2. Comparison with Saccharomyces cerevisiae Mediator expression data reveals that these functionally distinct modules are conserved between S. pombe and S. cerevisiae.
Assuntos
Subunidades Proteicas/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/genética , Transativadores/fisiologia , Alelos , Parede Celular/metabolismo , Quinase 8 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Perfilação da Expressão Gênica , Mutação , Fenótipo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Transativadores/química , Transativadores/genéticaRESUMO
The Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent genes. Here we demonstrate a new role for Mediator which appears to be separate from its function as a transcriptional coactivator. Mediator associates directly with heterochromatin at telomeres and influences the exact boundary between active and inactive chromatin. Loss of the Mediator Med5 subunit or mutations in Med7 cause a depletion of the complex from regions located near subtelomeric X elements, which leads to a change in the balance between the Sir2 and Sas2 proteins. These changes in turn result in increased levels of H4K16 acetylation near telomeres and in desilencing of subtelomeric genes. Increases in H4K16 acetylation have been observed at telomeres in aging cells. In agreement with this observation, we found that the loss of MED5 leads to shortening of the Saccharomyces cerevisiae (budding yeast) replicative life span.
Assuntos
Inativação Gênica , Complexo Mediador/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Senescência Celular/fisiologia , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Complexo Mediador/genética , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/genética , Sirtuína 2/metabolismoRESUMO
Med5 (Nut1) is identified here as a component of the Mediator tail region. Med5 is positioned peripherally to Med16 (Sin4) together with the three members of the putative Gal11 module, Med15 (Gal11), Med2, and Med3 (Pgd1). The biochemical analysis receives support from genetic interactions between med5delta and med15delta deletions. The med5delta and med16delta deletion strains share many phenotypes, including effects on mitochondrial function with enhanced growth on nonfermentable carbon sources, increased citrate synthase activity, and increased oxygen consumption. Deletion of the MED5 gene leads to increased transcription of nuclear genes encoding components of the oxidative phosphorylation machinery, whereas mitochondrial genes encoding components of the same machinery are down-regulated. We discuss a possible role for Med5 in coordinating nuclear and mitochondrial gene transcription.
Assuntos
Núcleo Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Transcrição Gênica , Animais , Carbono/química , Linhagem Celular , Cromatografia em Gel , Citrato (si)-Sintase/metabolismo , RNA Polimerases Dirigidas por DNA/química , Regulação para Baixo , Deleção de Genes , Immunoblotting , Insetos , Complexo Mediador , Mitocôndrias/metabolismo , Modelos Genéticos , Mutação , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oxigênio/química , Oxigênio/metabolismo , Consumo de Oxigênio , Peptídeos/química , Fenótipo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Temperatura , Fatores de Tempo , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro, and its function is conserved in all eukaryotes. Mediator interacts with both transcriptional activators and RNA polymerase II, but little is known about the mechanisms by which it operates at the molecular level. Here, we show that the cyclin-dependent kinase Srb10 interacts with, and phosphorylates, the Med2 subunit of Mediator both in vivo and in vitro. A point mutation of the single phosphorylation site in Med2 results in a strongly reduced expression of the REP1, REP2, FLP1, and RAF1 genes, which are all located on the endogenous 2-microm plasmid. Combined with previous studies on the effects of SRB10/SRB11 deletions, our data suggest that posttranslational modifications of Mediator subunits are important for regulation of gene expression.