Visualizing Genetic Activity in Normal & Mutant Yeast

描述（由申请人提供）：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组分，其合成速率决定核糖体生物合成的速率，而核糖体生物合成的速率又与细胞生长速率直接相关。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治疗药物部分通过减少RNA聚合酶I转录来发挥作用，并且正在测试专门针对这一过程的新药。在聚合酶本身的结构和功能分析以及参与制造核糖体RNA的其他因素方面已经取得了巨大的进展，但许多重要的问题仍然没有答案。Beyer实验室通过使用电子显微镜直接观察从生长细胞释放的活性rRNA基因，为这项研究工作提供了一种独特的体内方法。米勒染色质扩散方法允许高分辨率分析许多个体基因以及分析许多个体核仁中的基因群体。使用酿酒酵母作为模型系统，在存在和不存在关键调节蛋白（即，在适当的突变株），以确定这些蛋白质如何影响rRNA合成在活细胞中的个体基因水平。可以辨别和区分转录起始、延伸、终止、新生转录物的加工、染色质结构和模板拓扑结构中的变化或缺陷，以及这些过程之间的任何耦合。许多个别基因的定量分析允许确定统计学上显著的变化，并提供了机制的见解，如区分延伸因子，有助于识别的内在暂停网站和那些帮助聚合酶过境这样的网站。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）表征两种延伸因子Spt 5和FACT在rRNA转录和转录偶联RNA加工中的体内功能，和（3）研究RNA聚合酶I的转录终止，并鉴定来自新生转录物降解，内在暂停位点，蛋白质路障和聚合酶固有的RNA切割活性。通过活细胞活性基因的直接可视化获得的信息与其他研究者的遗传和生物化学分析相结合时是非常有价值的，并且一起提供了RNA聚合酶I对核糖体RNA合成的调节的更完整的画面。 公共卫生相关性：该项目的目标是帮助确定细胞如何控制核糖体的产生，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。该项目的重点是该过程的第一步--制造大的核糖体RNA--因为这一步控制着核糖体的制造速度。通过一种独特的方法获得的信息，涉及直接可视化的活性基因将揭示调控步骤，可能是有前途的新目标，在癌症治疗。

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.