Visualizing Genetic Activity in Normal & Mutant Yeast

描述(申请人提供)：拟议研究的长期目标是更好地了解调节核糖体RNA的RNA聚合酶I转录的分子机制。这种rRNA成为核糖体的主要结构和功能成分，其合成速度决定了核糖体生物发生的速度，而核糖体生物发生的速度又与细胞的生长速度直接相关。因此，了解这一过程对于了解癌细胞的生长特性具有重要意义。事实上，许多针对快速生长细胞的癌症疗法部分是通过减少RNA聚合酶I转录来发挥作用的，目前正在测试专门针对这一过程的新药。在对聚合酶本身以及参与制造核糖体RNA的其他因素的结构和功能分析方面已经取得了巨大的进展，但许多重要的问题仍然没有得到回答。拜尔实验室通过使用电子显微镜直接观察生长中细胞释放的活性rRNA基因，为这项研究工作贡献了一种独特的体内方法。米勒染色质扩散法允许对许多单个基因进行高分辨率分析，以及分析许多单个核仁中的基因群体。利用酿酒酵母作为模型系统，在存在和不存在关键调控蛋白的情况下(即在适当的突变菌株中)可视化rRNA基因，以确定这些蛋白如何在活细胞中的单个基因水平上影响rRNA合成。可以辨别和区分转录的起始、延伸、终止、新生转录本的处理、染色质结构和模板拓扑以及这些过程之间的任何耦合的变化或缺陷。对许多单个基因的定量分析可以确定统计上显著的变化，并提供机械性的洞察力，例如有助于识别固有停顿位点的伸长因子和帮助聚合酶运输这些位点的伸长因子之间的区别。本项目的具体目标是(1)表征RNA聚合酶I亚基在rRNA转录和转录偶联RNA加工中的体内功能，(2)表征两个延伸因子Spt5和FACT在rRNA转录和转录偶联RNA加工中的体内功能，以及(3)研究RNA聚合酶I的转录终止作用，并确定新生转录降解、内在暂停位点、蛋白质障碍和聚合酶固有的RNA切割活性的贡献。从活细胞中直接显示活性基因所获得的信息，如果与其他研究人员的遗传和生化分析相结合，将是非常有价值的，并共同提供了由RNA聚合酶I调节核糖体RNA合成的更完整的图景。 与公共卫生相关：该项目的目标是帮助确定细胞如何控制核糖体的生产，核糖体是细胞的蛋白质合成机器，因此是细胞快速生长所必需的，并在癌细胞中增加。该项目专注于这一过程的第一步--制造大的核糖体RNA--因为这一步控制着核糖体的制造速度。通过一种涉及活性基因直接可视化的独特方法获得的信息将揭示调控步骤，这些步骤可能是癌症治疗中有希望的新靶点。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to better understand the molecular mechanisms that regulate RNA polymerase I transcription of ribosomal RNA. This rRNA becomes the major structural and functional component of ribosomes and its rate of synthesis determines the rate of ribosome biogenesis, which in turn is directly correlated with cell growth rate. Thus it is of great importance to understand this process so as to understand the increased growth characteristic of cancer cells. Indeed, many cancer therapeutics that target rapidly growing cells function in part by decreasing RNA polymerase I transcription and new drugs are being tested that specifically target this process. Huge advances have been made in structural and functional analyses of the polymerase itself and of additional factors that participate in making ribosomal RNA, yet many important questions remain unanswered. The Beyer laboratory contributes a unique in vivo approach to this research effort by using electron microscopy to directly visualize active rRNA genes released from growing cells. The Miller chromatin spreading method allows high-resolution analysis of many individual genes as well as analysis of the gene population in many individual nucleoli. Using Saccharomyces cerevisiae as a model system, rRNA genes are visualized in the presence and absence of key regulatory proteins (i.e., in appropriate mutant strains) to determine how these proteins affect rRNA synthesis at the individual gene level in living cells. Changes or defects in transcription initiation, elongation, termination, processing of nascent transcripts, chromatin structure and template topology can be discerned and distinguished, as well as any coupling between these processes. Quantitative analysis of many individual genes allows determination of statistically significant changes and provides mechanistic insight, such as distinction between elongation factors that contribute to recognition of intrinsic pause sites and those that help polymerases transit such sites. The specific goals of this project are (1) to characterize the in vivo function of RNA polymerase I subunits in rRNA transcription and transcription-coupled RNA processing, (2) to characterize the in vivo function of two elongation factors, Spt5 and FACT, in rRNA transcription and transcription-coupled RNA processing, and (3) to investigate transcription termination by RNA polymerase I and identify contributions from nascent transcript degradation, intrinsic pause sites, protein roadblocks and RNA cleavage activity intrinsic to the polymerase. The information gained by direct visualization of active genes from living cells is extremely valuable when combined with genetic and biochemical analyses from other investigators, and together provide a much more complete picture of the regulation of ribosomal RNA synthesis by RNA polymerase I. PUBLIC HEALTH RELEVANCE: The goal of this project is to help determine how cells control the production of ribosomes, which are the cell's protein-synthesizing machines and thus are required for rapid cell growth and are increased in cancer cells. The project focuses on the first step in the process - making large ribosomal RNAs - because this step controls the rate at which ribosomes are made. Information gained by a unique approach that involves direct visualization of active genes will reveal regulatory steps that may be promising new targets in cancer therapy.