Genetics and Biochemistry of Ribosome Synthesis

描述（申请人提供）：核糖体是蛋白质合成所必需的中心机制，显然在细胞生长中起关键作用，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，核糖体合成的调节在肿瘤细胞的发展和生长中的重要作用。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。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合成的调节。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。先前的研究表明，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。在过去，我们集中在研究转录起始的机制在个别的活性基因。虽然我们将继续这方面的工作，现在将更多的重点放在研究后启动步骤和它的调控在个别活性基因。目前已知rRNA的修饰、加工和一些组装反应是在共转录状态下进行的。因此，rRNA的转录和加工/组装可能是偶联和共调节的。我们最近发现，Spt 4/5复合物（一种已知的mRNA转录延伸因子）中的突变缺陷导致Pol I延伸缺陷。我们计划确定rRNA基因内的区域，需要这个因子的转录和研究的作用，这个因子使用遗传和生物化学的方法。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。我们对那些在转录与rRNA加工/组装偶联方面存在缺陷的突变体特别感兴趣。关于这一目标，我们有未发表的观察结果显示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），然后研究所涉及的机制。最后，我们计划研究控制rRNA基因活性和非活性状态之间切换的机制。我们将比较与活性基因相关的RNA和蛋白质组分与与非活性基因相关的RNA和蛋白质组分，以建立对开关机制的理解。我们还计划分离开关缺陷的突变体，并研究这些突变体以确定其机制。我们期望所提出的工作将为我们理解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.