Genetics and Biochemistry of Ribosome Synthesis

描述(申请人提供)：核糖体，蛋白质合成的必要机械，显然在细胞生长中起着关键作用，其合成与原核和真核细胞中细胞增殖的调节密切相关。当细胞通过各种方式被刺激增殖时，核糖体合成的增加是在实际细胞增殖之前发生的非常早期的事件之一。我们的目标是了解细胞如何调节核糖体的生产，以应对环境条件。众所周知，核糖体RNA(RRNA)的合成是决定核糖体整体合成的核心，因此，我们将集中在rRNA合成的调控上。我们使用酿酒酵母作为一个模型系统，因为它易于遗传操作，并且已知RNA聚合酶I合成rRNA的知识。我们将继续研究RNA聚合酶I转录机制的组成部分与rDNA模板在体外和体内的相互作用。为了阐明调控机制，我们将分析这些成分和rDNA模板在具有高和低rRNA合成率的细胞中的状态。为此，将使用染色质免疫沉淀试验、提取物的体外rDNA转录试验和rDNA染色质铺展的电子显微镜分析。还将使用遗传方法来确定监管中涉及的成分。RRNA的基因在核仁中随机重复，核仁在细胞核中占据与其他染色体基因分开的特定区域。这反映在RNA聚合酶II报告基因的沉默现象上，我们将从rDNA基因的状态来研究它。核仁作为核糖体合成的场所，显然对细胞生长具有重要作用，但其结构复杂，其功能重要性可能尚未得到充分认识。事实上，最近的研究表明，它在细胞周期调节中的重要性，可能在细胞衰老中也是如此。我们将使用遗传学方法来研究rDNA基因的拷贝数、rDNA中的DNA元件以及rDNA基因的染色体位置在核仁结构和功能中的重要性。我们期望拟议的工作将对我们理解重要的核仁功能做出重大贡献，包括核糖体的合成及其调控。

DESCRIPTION (provided by applicant): The ribosome, the machinery essential for protein synthesis, obviously plays a key role in cell growth and its synthesis is intimately connected to the regulation of cell proliferation both in prokaryotic and eukaryotic cells. When cells are stimulated to proliferate by various means, an increase in ribosome synthesis takes place as one of the very early events before actual cell proliferation occurs. Our goal is to understand how cells regulate the production of ribosomes in response to environmental conditions. It is known that synthesis of ribosomal RNA (rRNA) is central in determining overall synthesis of ribosomes, and hence, we will concentrate on regulation of rRNA synthesis. We use the yeast Saccharomyces cerevisiae as a model system because of the ease of genetic manipulation and the available knowledge of rRNA synthesis by RNA polymerase I. We will continue to study interactions of components of the RNA polymerase I transcription machinery with the rDNA template both in vitro and in vivo. To elucidate the mechanisms of regulation, we will analyze the state of these components and the rDNA template in cells with high and low rRNA synthesis rates. Chromatin immunoprecipitation assay, in vitro rDNA transcription assay of extracts and electron microscopic analysis of rDNA chromatin spreads will be used for this purpose. Genetic approaches will also be used to identify components involved in the regulation. The genes for rRNAs are tandemly repeated in the nucleolus, which occupies a specific region in the nucleus separate from other chromosomal genes. This is reflected in the phenomenon of silencing of reporter genes for RNA polymerase II, and we will study it in relation to the state of rDNA genes. The nucleolus is obviously important for cell growth as the site of ribosome synthesis, but its structure is complex and its functional importance may not be fully appreciated. In fact, recent studies suggest its importance in regulation of cell cycle and possibly in cell aging. We will use genetic approaches to examine the importance of the copy number of rDNA genes, DNA elements within rDNA and chromosomal position of rDNA genes in nucleolar structure and function. We expect that the proposed work will make significant contributions to our understanding of important nucleolar functions including ribosome synthesis and its regulation.