GTPase control of large ribosome subunit biogenesis

描述(由申请人提供)： 项目总结。核糖体是由3个RNA和50多种蛋白质组成的复杂的分子纳米机器，负责将信使核糖核酸解码成蛋白质。最近发表的几种核糖体结构，与它们的底物、辅因子和抗生素形成了复合体，具有 极大地增加了我们对核糖体在翻译过程中功能的理解。然而，从对完全组装的核糖体的检查中可以提取的关于核糖体生物发生的信息很少。阻碍细菌核糖体组装研究的一个主要挑战来自这样一个事实，即这个过程在体内是高效的，而且不成熟的中间产物不会积累。我们发现，辅助核糖体组装的因子是研究核糖体成熟过程的关键工具。针对这些因子的突变减缓了核糖体的组装，并使分离和鉴定核糖体中间体成为可能。最近对必需GTP酶在核糖体组装中的作用的研究表明，在生命的三个领域中，30s和50s的组装都需要几种GTP酶。我们实验室以前的工作已经证明，RbgA、YphC和YsxC是参与枯草杆菌核糖体大亚基在体内成熟的GTP酶。尽管细菌、古生物和真核生物中的几种GTP酶已被确定参与核糖体组装，但这些蛋白在这一过程中所起的确切作用仍然是个谜。遗传、生化和结构数据支持一种模型，在该模型中，RbgA在协调参与后期组装的核糖体蛋白的掺入方面发挥重要作用，并协调中央突起以及A、P和E tRNA结合位点的形成。更多的遗传和生化证据表明，必需的GTP酶YphC(EngA)和YsxC也参与了50s亚基的后期成熟。我们的工作假设是，RbgA、YphC和YsxC通过识别共同的晚期组装中间体起协同作用，它们与该中间体结合并催化50s组装的最后步骤。或者，它们可以顺序作用于不同的大亚基中间体。为了进一步了解这些GTP酶如何帮助大亚基的组装，提出了以下具体目标：目的1.鉴定和比较RbgA、YphC和YsxC耗竭细胞的未成熟中间体。目的2.建立RbgA、YphC和YsxC与装配50S亚基结合的功能层次结构。目的3.确定RbgA、YphC和YsxC在核糖体上的结合部位。我们的三个研究小组，跨越遗传学、生物化学、定量质谱学和冷冻电子显微镜的协同方法，为我们在这个项目中取得成功奠定了基础。我们预计这项工作将对理解50s亚基成熟产生深远影响，并对细菌核糖体生物发生过程的可药性和新抗菌剂的开发产生影响。

DESCRIPTION (provided by applicant): Project summary. Ribosomes, responsible for decoding mRNA into proteins, are complex molecular nanomachines made up of three RNAs and over 50 proteins. The recent publication of several ribosome structures, in complex with their substrates, co-factors, and antibiotics, have greatly increased our understanding of how the ribosome functions during translation. However, very little information about ribosome biogenesis can be extracted from the inspection of the fully assembled ribosome. A major challenge hampering the study of ribosome assembly in bacteria comes from the fact that the process is highly efficient in vivo and immature intermediates do not accumulate. We have found that factors assisting ribosomal assembly represent key tools to study the process of ribosome maturation. Mutations directed at these factors slow down ribosome assembly and make it possible to isolate and characterize ribosomal intermediates. Recent studies investigating the roles of essential GTPases in ribosome assembly have shown that several GTPases are required for both 30S and 50S assembly in all three domains of life. Previous work from our laboratory has demonstrated that RbgA, YphC and YsxC are GTPases that participate in the maturation of the large ribosomal subunit in vivo in Bacillus subtilis. Although several GTPases in bacteria, archaea, and eukaryotes have been identified as participating in ribosome assembly, the precise role these proteins play in this process remains a mystery. Genetic, biochemical, and structural data support a model in which RbgA plays an essential role in coordinating the incorporation of ribosomal proteins involved in late assembly and coordinates the formation of the central protuberance and the A, P and E tRNA binding sites. Additional genetic and biochemical evidence suggests the essential GTPases YphC (EngA) and YsxC also participate in the late stages of maturation of the 50S subunit. Our working hypothesis posits that RbgA, YphC, and YsxC act in conjunction by recognizing a common late assembly intermediate to which they bind and catalyze the final steps of 50S assembly. Alternatively, they may act sequentially on distinct large subunit intermediates. To further understand how these GTPases assist the assembly of the large subunit the following specific aims are proposed: Aim 1. Characterize and compare immature intermediates from RbgA, YphC and YsxC-depleted cells. Aim 2. Establish the functional hierarchy of RbgA, YphC and YsxC binding to the assembling 50S subunit. Aim 3. Define the binding sites of RbgA, YphC and YsxC on the ribosome. The synergistic approaches of our three research groups, spanning genetics, biochemistry, quantitative mass spectrometry and cryo-electron microscopy position us for success in this project. We anticipate this work will have profound impact on the understanding of 50S subunit maturation with implications for the druggability of the bacterial ribosome biogenesis process and the development of new antimicrobials.