Genetics and Biochemistry of Ribosome Synthesis

描述（由申请人提供）：核糖体是蛋白质合成所必需的机器，显然在细胞生长中起关键作用，并且其合成与原核和真核细胞中细胞增殖的调节密切相关。当通过各种方式刺激细胞增殖时，核糖体合成的增加作为实际细胞增殖发生之前的非常早期的事件之一而发生。我们的目标是了解细胞如何调节核糖体的产生以应对环境条件。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，我们将集中于rRNA合成的调控。我们使用酵母酿酒酵母作为模型系统，因为易于遗传操作和RNA聚合酶I合成rRNA的可用知识。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，我们将分析这些组分的状态和rDNA模板在细胞中的高和低rRNA合成速率。染色质免疫沉淀试验、提取物的体外rDNA转录试验和rDNA染色质扩散的电子显微镜分析将用于此目的。遗传学方法也将用于确定参与调控的成分。rRNA基因在核仁中串联重复，核仁占据了细胞核中与其他染色体基因分开的特定区域。这反映在RNA聚合酶II的报告基因沉默的现象中，我们将研究它与rDNA基因状态的关系。作为核糖体合成的场所，核仁对于细胞生长显然是重要的，但是其结构复杂并且其功能重要性可能未被充分认识。事实上，最近的研究表明它在细胞周期调控中的重要性，并可能在细胞衰老中发挥作用。我们将使用遗传学方法来研究rDNA基因的拷贝数，rDNA内的DNA元件和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，拟议的工作将作出重大贡献，我们了解重要的核仁功能，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.