In vivo Assembly of the Large Ribosomal Subunit

大核糖体亚基的体内组装

• 批准号: 7368515
• 负责人: Janine R. Maddock
• 金额: $ 25.15万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-10 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7368515
• 关键词: Affinity; Antibiotics; Bacteria; Binding; Biogenesis; Cells; Complex; Event; Gene Proteins; Generations; Genes; Genetic; Goals; Hand; In Vitro; Lead; Macromolecular Complexes; Mitochondria; Modification; Molecular Structure; Numbers; Pathway interactions; Process; Proteins; Proteomics; Reagent; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Saccharomyces cerevisiae; Spottings; Structure; Technology; Testing; antimicrobial; cell type; design; human disease; in vivo; mutant; particle; reconstitution; research study; tool

DESCRIPTION (provided by applicant): Assembly of the large bacterial ribosomal subunit (50S) requires the coordinated synthesis of two rRNAs (5S and 23S) and 33 ribosomal proteins (r-proteins), processing and modification of the rRNAs, and assembly of both rRNAs and r-proteins into the functional subunit. Although assembly of the 50S subunit can be reconstituted in vitro, ribosome biogenesis in vivo has not been described. Since in vivo ribosome assembly also requires the activities of a number of ribosome assembly factors, determining when these assembly factors bind and defining the pre-50S particles that accumulate in their absence is critical for understanding in vivo assembly. Our long-term goal is to delineate the cellular events that lead to the generation of the 50S subunit. We are particularly focused on solving the temporal changes that occur during 50S biogenesis and the roles of ribosome assembly factors. We are now well poised to achieve this goal having recently made significant advances in defining the in vivo 50S particle using advanced proteomic approaches. Moreover, we have successfully used cutting edge proteomics to analyze one pre-50S intermediate. We have in hand a large number of verified ribosome-associated proteins and ribosome-assembly factors. We also have the genetic reagents (mutants, cloned genes and protein fusions) to define the assembly pathway for this complicated macromolecular structure. In this proposal, we will provide detailed characterization of assembly intermediates, isolate additional putative assembly factors and define functional relationships between ribosome-associated proteins and r-proteins. In Aim 1, we will define the in vivo assembly pathway for the 50S ribosomal subunit. To do this, we will build upon existing tools and reagents, guided in large part by the elegant ribosome intermediate studies being performed in S. cerevisiae. Our approach will be to purify intermediates in ribosome assembly and characterize both the rRNA and proteins. In these studies, we will apply proteomic approaches (iTRAQ quantitation technology) to define ribosome assembly intermediates. In addition to characterizing particles affinity purified pre-50S complexes from wild type cells, we will define intermediates that accumulate in a variety of ribosome assembly mutants. New proteins identified in this study will be assessed for their involvement in 50S assembly. In Aim 2, we will identify heretofore-unknown relationships by isolating high copy suppressors of existing ribosome assembly mutants. We will also define the relationship between the suppressor and the suppressed gene at the level of ribosome assembly and also in regards to testing specific predictions that we will develop for each suppressed mutant-suppressor pair. Experiments to test specific hypotheses concerning existing genetically interacting proteins are proposed. Project Narrative: Ribosomes are the largest macromolecular complex in the cell and, in bacteria, are the target of many antibiotics. Although the atomic structure of ribosomes has been solved and an in vitro assembly pathway defined, we know little about how ribosomes are assembled inside of bacterial cells. The goal of this proposal is to define the in vivo assembly pathway of the bacterial large ribosomal subunit, a feat that will have significant implications on the design of new antimicrobials and on understanding human diseases associated with the conserved mitochondrial ribosomes.

描述（由申请人提供）：大型细菌核糖体亚基（50s）的组装要求将两个RRNA（5s和23s）和33个核糖体蛋白（R蛋白）（R蛋白），RRNA的加工和修饰以及RRNAS和RRNAS的组装进行协调合成。尽管可以在体外重构50S亚基的组装，但尚未描述体内核糖体生物发生。由于体内核糖体组装也需要许多核糖体组装因子的活性，因此确定这些组装因子何时结合并定义在缺乏其缺失的50前颗粒中，对于理解体内组装中的理解至关重要。我们的长期目标是描述导致50年代亚基产生的细胞事件。我们尤其专注于解决在50年代生物发生和核糖体组装因子的作用期间发生的时间变化。现在，我们已经准备好实现这一目标，最近使用先进的蛋白质组学方法在定义体内50S粒子方面取得了重大进展。此外，我们已经成功地使用了尖端蛋白质组学来分析一个50前的中间体。我们手头有大量经过验证的核糖体相关蛋白和核糖体组装因子。我们还具有遗传试剂（突变体，克隆基因和蛋白质融合），以定义这种复杂的大分子结构的组装途径。在此提案中，我们将提供组装中间体的详细表征，隔离其他推定的装配因子，并定义与核糖体相关蛋白和R蛋白之间的功能关系。在AIM 1中，我们将定义50S核糖体亚基的体内组装途径。为此，我们将建立在现有工具和试剂的基础上，这在很大程度上是由在酿酒酵母中进行的优雅核糖体中间研究引导的。我们的方法是纯化核糖体组装中的中间体，并表征rRNA和蛋白质。在这些研究中，我们将应用蛋白质组学方法（ITRAQ定量技术）来定义核糖体组装中间体。除了表征来自野生型细胞的颗粒亲和力纯化的50s复合物外，我们还将定义在各种核糖体组装突变体中积聚的中间体。将评估本研究中确定的新蛋白质，以评估其参与50S组装。在AIM 2中，我们将通过隔离现有核糖体组装突变体的高拷贝抑制器来确定迄今未知的关系。我们还将在核糖体组装水平上定义抑制剂与抑制基因之间的关系，以及在测试每个抑制突变抑制剂对的特定预测方面。提出了测试有关现有遗传相互作用蛋白的特定假设的实验。 项目叙述：核糖体是细胞中最大的大分子复合物，在细菌中，核糖体是许多抗生素的靶标。尽管核糖体的原子结构已被溶解并定义了体外组装途径，但我们对核糖体如何在细菌细胞内部组装在一起一无所知。该建议的目的是定义细菌大核糖体亚基的体内组装途径，这一壮举将对新抗微生物的设计以及与保守线粒体核糖体相关的人类疾病的设计具有重要意义。