GTPase control of large ribosome subunit biogenesis

描述（由申请人提供）： 项目摘要。核糖体负责将mRNA解码成蛋白质，是由三种RNA和50多种蛋白质组成的复杂分子纳米机器。最近发表的几种核糖体结构，与它们的底物、辅因子和抗生素复合， 大大增加了我们对核糖体在翻译过程中如何发挥作用的理解。然而，从完全组装的核糖体的检查中可以提取很少的关于核糖体生物发生的信息。阻碍细菌中核糖体组装研究的一个主要挑战来自这样一个事实，即该过程在体内非常有效，不成熟的中间体不会积累。我们已经发现，有助于核糖体组装的因素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核糖体组装，并使分离和表征核糖体中间体成为可能。最近的研究调查的作用，在核糖体组装的基本GTP酶已经表明，几个GTP酶都需要为30 S和50 S组装在所有三个领域的生活。我们实验室的前期工作已经证明RbgA、YphC和YsxC是参与枯草芽孢杆菌体内核糖体大亚基成熟的GTP酶。尽管细菌、古细菌和真核生物中的几种GTP酶已被鉴定为参与核糖体组装，但这些蛋白质在此过程中发挥的确切作用仍然是一个谜。遗传，生物化学和结构数据支持的模型中，RbgA起着重要的作用，在协调参与后期组装的核糖体蛋白的掺入和协调的中央突起和A，P和E tRNA结合位点的形成。另外的遗传和生物化学证据表明，必需的GTP酶YphC（EngA）和YsxC也参与50S亚基成熟的晚期阶段。我们的工作假设假定RbgA，YphC和YsxC通过识别它们结合并催化50S组装的最后步骤的共同后期组装中间体而共同起作用。或者，它们可以依次作用于不同的大亚基中间体。为了进一步了解这些GTP酶如何协助大亚基的组装，提出了以下具体目标：目标1。表征并比较来自RbgA、YphC和YsxC耗尽细胞的未成熟中间体。目标2.建立RbgA、YphC和YsxC与组装50S亚基结合的功能层次。目标3：确定核糖体上RbgA、YphC和YsxC的结合位点。我们三个研究小组的协同方法，跨越遗传学，生物化学，定量质谱和冷冻电子显微镜定位我们在这个项目中取得成功。我们预计这项工作将对理解50S亚基成熟产生深远的影响，并对细菌核糖体生物合成过程的可药用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.