Genetics and Biochemistry of Ribosome Synthesis

描述(申请人提供)：核糖体，蛋白质合成必不可少的中央机械，显然在细胞生长中起着关键作用，其合成与细胞增殖的调节密切相关。事实上，最近的研究表明，核糖体合成在肿瘤细胞的发育和生长中起着重要的调节作用。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。由于核糖体RNA(RRNA)的转录是决定核糖体整体合成的核心，我们的工作集中在RNA聚合酶I(Pol I)对rRNA合成的调控上。我们使用酿酒酵母作为模型系统，是因为它易于遗传操作，而且对RNA聚合酶I的rRNA合成有了解。以往的研究表明，酵母细胞通过两种机制改变rRNA合成速率。一种是通过改变活性(开放)基因的数量，另一种是通过改变单个活性基因的活性。过去，我们主要研究单个活性基因的转录启动机制。虽然我们将继续这项工作，但现在将更加重视启动后步骤及其对单个活性基因的调控的研究。现在已经知道，rRNA的修饰、加工和一些组装反应是通过共转录发生的。因此，rRNA的转录和加工/组装很可能是耦合和共同调节的。我们最近发现了Spt4/5复合体的突变缺陷，Spt4/5复合体是一种已知的mRNA转录延伸因子，它导致了POL I的延伸缺陷。我们计划找出rRNA基因中需要该因子转录的区域，并利用遗传学和生化方法研究该因子所起的作用。我们将分离在Pol I转录延伸步骤中具有特定缺陷的突变体。我们特别感兴趣的是那些在转录与rRNA加工/组装的耦合方面存在缺陷的突变体。关于这一目标，我们有未发表的观察结果表明，18S rRNA合成(和/或40S亚基组装)明显依赖于5.8S-25S rRNA合成(和/或60S亚基组装)。我们计划确定18S rRNA转录本身，而不是40S亚基组装，是否依赖于5.8S-25S rRNA转录(和/或40S亚基组装)，然后研究涉及的机制。最后，我们计划研究控制rRNA基因在激活和非激活状态之间切换的机制。我们将比较与活性基因和非活性基因相关的RNA和蛋白质成分，以建立对涉及转换的机制的理解。我们还计划分离开关缺陷突变株，并对这些突变株进行研究，以确定其机制。我们期待所提出的工作将对我们理解rRNA基因的转录及其调控做出重大贡献。

DESCRIPTION (provided by applicant): The ribosome, the central 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. Indeed, recent studies suggest important roles for regulation of ribosome synthesis in the development and growth of tumor cells. Our goal is to understand how cells regulate the production of ribosomes in response to environmental conditions. Since transcription of ribosomal RNA (rRNA) is central in determining overall synthesis of ribosomes, our work focuses on regulation of rRNA synthesis by RNA Polymerase I (Pol I). 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. Previous studies showed that yeast cells change rRNA synthesis rate by two mechanisms. One is by altering the number of active (open) genes, and another is by altering the activity of individual active genes. In the past, we concentrated on studies of mechanisms of initiation of transcription at individual active genes. Although we will continue this line of work, more emphasis will now be given to studies of post-initiation steps and its regulation at individual active genes. It is now known that rRNA modification, processing and some of assembly reactions take place co-transcriptionally. Therefore, it is likely that transcription and processing/assembly of rRNA are coupled and co-regulated. We have recently identified that mutational defects in the Spt4/5 complex, a known elongation factor for mRNA transcription, cause defects in elongation of Pol I. We plan to identify regions within the rRNA gene that require this factor for their transcription and study roles played by this factor using genetic and biochemical approaches. We will isolate mutants with specific defects in Pol I transcription elongation steps. We are especially interested in those mutants with defects in coupling of transcription with rRNA processing/assembly. In relation to this goal, we have unpublished observations showing an apparent dependence of 18S rRNA synthesis (and/or 40S subunit assembly) on 5.8S-25S rRNA synthesis (and/or 60S subunit assembly). We plan to determine whether 18S rRNA transcription itself, rather than 40S subunit assembly, is dependent on 5.8S-25S rRNA transcription (and/or 40S subunit assembly), and then to study the mechanism involved. Finally, we plan to study the mechanism that controls switching between active and inactive states of rRNA genes. We will compare RNA and protein components associated with active genes with those associated with inactive genes in order to build an understanding of the mechanism involved in switching. We also plan to isolate mutants defective in switching and study these mutants to identify the mechanism. We expect that the proposed work will make significant contributions to our understanding of transcription of rRNA genes and its regulation.