RESUMEN
Nutrient deprivation and various stress conditions repress RNA polymerase III (Pol III) transcription in S. cerevisiae. The signaling pathways that relay stress and nutrient conditions converge on the conserved protein Maf1, but how Maf1 integrates environmental conditions and couples them to transcriptional repression is largely unknown. Here, we demonstrate that Maf1 is phosphorylated in favorable conditions, whereas diverse unfavorable conditions lead to rapid Maf1 dephosphorylation, nuclear localization, physical association of dephosphorylated Maf1 with Pol III, and Maf1 targeting to Pol III-transcribed genes genome wide. Furthermore, Maf1 mutants defective in full dephosphorylation display maf1Delta phenotypes and are compromised for both nuclear localization and Pol III association. Repression conditions also promote TFIIIB-TFIIIC interactions in crosslinked chromatin. Taken together, Maf1 appears to integrate environmental conditions and signaling pathways through its phosphorylation state, with stress leading to dephosphorylation, association with Pol III at target loci, alterations in basal factor interactions, and transcriptional repression.
Asunto(s)
ARN Polimerasa III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Núcleo Celular/metabolismo , Represión Enzimática/genética , Represión Enzimática/fisiología , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Mutación Puntual , ARN Polimerasa III/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
RNA polymerase III (Pol III) transcribes small untranslated RNAs, such as tRNAs. To define the Pol III transcriptome in Saccharomyces cerevisiae, we performed genome-wide chromatin immunoprecipitation using subunits of Pol III, TFIIIB and TFIIIC. Virtually all of the predicted targets of Pol III, as well as several novel candidates, were occupied by Pol III machinery. Interestingly, TATA box-binding protein occupancy was greater at Pol III targets than virtually all Pol II targets, and the highly occupied Pol II targets are generally strongly transcribed. The temporal relationships between factor occupancy and gene activity were then investigated at selected targets. Nutrient deprivation rapidly reduced both Pol III transcription and Pol III occupancy of both a tRNA gene and RPR1. In contrast, TFIIIB remained bound, suggesting that TFIIIB release is not a critical aspect of the onset of repression. Remarkably, TFIIIC occupancy increased dramatically during repression. Nutrient addition generally reestablished transcription and initial occupancy levels. Our results are consistent with active Pol III displacing TFIIIC, and with inactivation/release of Pol III enabling TFIIIC to bind, marking targets for later activation. These studies reveal new aspects of the kinetics, dynamics, and targets of the Pol III system.