RESUMO
Nuclear eukaryotic RNA polymerases (RNAPs) transcribe a chromatin template in vivo. Since the basic unit of chromatin, the nucleosome, renders the DNA largely inaccessible, RNAPs have to overcome the nucleosomal barrier for efficient RNA synthesis. Gaining mechanistical insights in the transcription of chromatin templates will be essential to understand the complex process of eukaryotic gene expression. In this article we describe the use of defined in vitro transcription systems for comparative analysis of highly purified RNAPs I-III from S. cerevisiae (hereafter called yeast) transcribing in vitro reconstituted nucleosomal templates. We also provide a protocol to study promoter-dependent RNAP I transcription of purified native 35S ribosomal RNA (rRNA) gene chromatin.
Assuntos
Nucleossomos , Saccharomyces cerevisiae , Cromatina/genética , Cromatina/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Moldes Genéticos , Transcrição GênicaRESUMO
In growing eukaryotic cells, nuclear ribosomal (r)RNA synthesis by RNA polymerase (RNAP) I accounts for the vast majority of cellular transcription. This high output is achieved by the presence of multiple copies of rRNA genes in eukaryotic genomes transcribed at a high rate. In contrast to most of the other transcribed genomic loci, actively transcribed rRNA genes are largely devoid of nucleosomes adapting a characteristic "open" chromatin state, whereas a significant fraction of rRNA genes resides in a transcriptionally inactive nucleosomal "closed" chromatin state. Here, we review our current knowledge about the nature of open rRNA gene chromatin and discuss how this state may be established.
Assuntos
Cromatina , Eucariotos , Cromatina/genética , DNA Ribossômico/genética , Eucariotos/genética , Eucariotos/metabolismo , Genes de RNAr , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Ribossômico/genética , Transcrição GênicaRESUMO
RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA) in all eukaryotes, accounting for the major part of transcriptional activity in proliferating cells. Although basal Pol I transcription factors have been characterized in diverse organisms, the molecular basis of the robust rRNA production in vivo remains largely unknown. In S. cerevisiae, the multifunctional Net1 protein was reported to stimulate Pol I transcription. We found that the Pol I-stimulating function can be attributed to the very C-terminal region (CTR) of Net1. The CTR was required for normal cell growth and Pol I recruitment to rRNA genes in vivo and sufficient to promote Pol I transcription in vitro. Similarity with the acidic tail region of mammalian Pol I transcription factor UBF, which could partly functionally substitute for the CTR, suggests conserved roles for CTR-like domains in Pol I transcription from yeast to human.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , RNA Polimerase I/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Sequência Conservada , Humanos , Proteínas Nucleares/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/química , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Transcrição GênicaRESUMO
Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p-Noc1p-Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity.
