RESUMEN
Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although Spt6 is viewed as an elongation factor, spt6 mutations in Saccharomyces cerevisiae allow elevated levels of transcripts from within coding regions, suggesting that Spt6 also controls initiation. To address the requirements for Spt6 in transcription and chromatin structure, we have combined four genome-wide approaches. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters and find sequence features conserved with genic promoters. Finally, we show that Spt6 also regulates transcription initiation at most genic promoters and propose a model of initiation site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome.
Asunto(s)
Chaperonas de Histonas/genética , Chaperonas de Histonas/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Iniciación de la Transcripción Genética/fisiología , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/fisiología , Cromatina/fisiología , Regulación Fúngica de la Expresión Génica/genética , Chaperonas de Histonas/metabolismo , Histonas/fisiología , Proteínas Nucleares , Nucleosomas , Factores de Elongación de Péptidos/fisiología , Regiones Promotoras Genéticas/genética , ARN Polimerasa II , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiología , Factores de Transcripción/fisiología , Sitio de Iniciación de la Transcripción/fisiología , Transcripción Genética/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
Comparative analysis of ribosomal RNA (rRNA) sequences has elucidated phylogenetic relationships. However, this powerful approach has not been fully exploited to address ribosome function. Here we identify stretches of evolutionarily conserved sequences, which correspond with regions of high functional importance. For this, we developed a structurally aligned database, FLORA (full-length organismal rRNA alignment) to identify highly conserved nucleotide elements (CNEs) in 23S-28S rRNA from each phylogenetic domain (Eukarya, Bacteria, and Archaea). Universal CNEs (uCNEs) are conserved in sequence and structural position in all three domains. Those in regions known to be essential for translation validate our approach. Importantly, some uCNEs reside in areas of unknown function, thus identifying novel sequences of likely great importance. In contrast to uCNEs, domain-specific CNEs (dsCNEs) are conserved in just one phylogenetic domain. This is the first report of conserved sequence elements in rRNA that are domain-specific; they are largely a eukaryotic phenomenon. The locations of the eukaryotic dsCNEs within the structure of the ribosome suggest they may function in nascent polypeptide transit through the ribosome tunnel and in tRNA exit from the ribosome. Our findings provide insights and a resource for ribosome function studies.
Asunto(s)
Biología Computacional , Filogenia , ARN Ribosómico/genética , ADN Ribosómico/genética , ARN Ribosómico/clasificación , Alineación de SecuenciaRESUMEN
Protein-only (prion) epigenetic elements confer unique phenotypes by adopting alternate conformations that specify new traits. Given the conformational flexibility of prion proteins, protein-only inheritance requires efficient self-replication of the underlying conformation. To explore the cellular regulation of conformational self-replication and its phenotypic effects, we analyzed genetic interactions between [PSI(+)], a prion form of the S. cerevisiae Sup35 protein (Sup35([PSI+])), and the three N(alpha)-acetyltransferases, NatA, NatB, and NatC, which collectively modify approximately 50% of yeast proteins. Although prion propagation proceeds normally in the absence of NatB or NatC, the [PSI(+)] phenotype is reversed in strains lacking NatA. Despite this change in phenotype, [PSI(+)] NatA mutants continue to propagate heritable Sup35([PSI+]). This uncoupling of protein state and phenotype does not arise through a decrease in the number or activity of prion templates (propagons) or through an increase in soluble Sup35. Rather, NatA null strains are specifically impaired in establishing the translation termination defect that normally accompanies Sup35 incorporation into prion complexes. The NatA effect cannot be explained by the modification of known components of the [PSI(+)] prion cycle including Sup35; thus, novel acetylated cellular factors must act to establish and maintain the tight link between Sup35([PSI+]) complexes and their phenotypic effects.