RESUMO
We report genome-wide analyses that establish Maf1 as a general and direct repressor of yeast RNA polymerase (Pol) III transcription. Chromatin immunoprecipitation (ChIP) coupled to microarray hybridization experiments showed an increased association of Maf1 to Pol III-transcribed genes under repressing condition (rapamycin treatment) correlated with a dissociation of Brf1 and Pol III. Maf1 can exist in various phosphorylation states and interacts with Pol III in a dephosphorylated state. The largest subunit of Pol III, C160, was identified as a target of Maf1. Under repressing conditions, Maf1 is dephosphorylated and accumulates in the nucleus, and Pol III-Maf1 interaction increases. Mutations in protein phosphatase type 2A (PP2A) catalytic subunit-encoding genes prevented rapamycin-induced Maf1 dephosphorylation, its nuclear accumulation, and repression of Pol III transcription. The results indicate that Pol III transcription can be globally and rapidly downregulated via dephosphorylation and relocation of a general negative cofactor.
Assuntos
Regulação Fúngica da Expressão Gênica , Fosfoproteínas Fosfatases/metabolismo , RNA Polimerase III/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Regulação para Baixo , Repressão Enzimática/genética , Sinais de Localização Nuclear/metabolismo , Fosforilação , Subunidades Proteicas/metabolismo , RNA Polimerase III/metabolismo , Transdução de Sinais , Sirolimo/metabolismo , Sirolimo/farmacologia , Fator de Transcrição TFIIIB/metabolismoRESUMO
Maf1p is a negative effector of RNA polymerase III in yeast. The maf1-delta mutation caused an increase in the level of cellular tRNAs, but a decrease of translational readthrough at nonsense codons. Using the lacZ- luc dual gene reporter system, we detected an almost twofold diminution of UAA and UAG readthrough in maf1-delta compared with the parental strain. The maf1-delta mutation did not affect the rate of protein biosynthesis and growth at standard conditions, but resulted in temperature-sensitive growth on non-fermentable carbon sources. We examined the correlation of the temperature sensitive and antisuppression phenotypes of maf1- Delta using a colour phenotype assay in the ade2-1 SUP11 strain. Antisuppression, but not the temperature-sensitive growth defect, was compensated either by increased dosage of SUP11or by [PSI(+)], the prion form of the translation termination factor Sup35p. Summarizing, the elevated tRNA levels in maf1- Delta increase translational fidelity and, independently, affect growth under special conditions.
Assuntos
Mutação , Biossíntese de Proteínas , RNA de Transferência/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Códon sem Sentido , Códon de Terminação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fatores de Terminação de Peptídeos , Fenótipo , Príons/genética , Príons/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Supressão Genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
tRNA isopentenylation is a branch of an isoprenoid pathway in yeast. There is a competition for a substrate between isoprenoid biosynthetic enzyme Erg20p and tRNA isopentenyltransferase. Here we studied the direct effect of elevated tRNA biosynthesis on ERG20 expression. The maf1-1 mutant of Saccharomyces cerevisiae that has enhanced cellular tRNA levels was used. We show that both ERG20 transcript and Erg20 protein levels are increased in maf1-1. Additionally, maf1-1 leads to decreased ergosterol content in the cells. These effects of maf1-1 are dependent on functional tRNA isopentenyltransferase. Our results indicate that a complex regulation of the isoprenoid pathway involves also an effect of changes in tRNA biosynthesis.
