Understanding ribosome biogenesis

了解核糖体的生物发生我的研究计划的总体目标是了解细菌如何生长和生存。在拟议的研究中，我们将集中在最基本的问题上，以了解核糖体是如何组装的。大肠杆菌核糖体是由一个16S核糖体RNA和21个30S亚基核糖体蛋白，以及一个5S rRNA、一个23S rRNA和34个50S亚基蛋白组装而成。野村实验室40多年前的研究表明，核糖体亚基可以在试管中与其组成的核糖体rna和蛋白质组装在一起。这些和更多的当代研究大大提高了我们对塑造亚单位组装的力量的理解。然而，这些实验是在非生理条件和时间下进行的，值得注意的是，大肠杆菌组装核糖体的速度非常快——大约在两分钟内——而且它是用50多种非核糖体蛋白因子组装的。在体内研究核糖体的组装是很困难的，因为组装中间体不会大量积累，无法进行分离。然而，协助核糖体组装的因素代表了核糖体成熟的非凡探针。针对这些因子的突变可以使核糖体的生物发生失效，并使分离和表征扰动产物成为可能。这种方法促进了对折叠和组装的微妙方面的具有挑战性的研究，这是本提案的重点。最有趣的是，一组新兴的生物发生蛋白，所谓的“神秘因子”，是gtp酶和rna结合蛋白，似乎在组装过程的后期步骤中具有伴侣或检查点作用。迄今为止，我们的大部分工作都集中在YjeQ蛋白上，并揭示了它在30S亚基组装后期的作用。越来越多的证据表明，几个生物发生因子构成了一个功能网络，并阻止晚期组装中间体过早进入70S核糖体的翻译池。对于30S亚基，目前的重点是一系列神秘的因子，包括BipA、Era、RbfA、RimM、RimP和YjeQ。在我的实验室进行的一项有限的遗传相互作用研究显示，Era（一种被认为在30S组装中起作用的GTPase）的过表达抑制了yjeQ缺失菌株的缓慢生长和核糖体缺陷。同样，失去神秘的30S组装因子RimM导致这些表型的增强。在本文提出的研究中，除了进一步研究30S核糖体亚基组装的意义外，我们还将扩大我们的努力，绘制这些神秘因素的功能相互作用的全基因组图谱。我们的短期目标如下：探索30S生物发生中未知因子的功能网络。目标2。检验由神秘因素的功能网络所提出的假设。目标3。当越来越多的人认为核糖体的生物发生是通过多个平行的途径进行的，这些途径是由可有可无和冗余的组装因子引导的，所提出的系统方法将是取得有效进展的关键。我的团队在高通量生物学和传统生物化学方面的独特能力将使我们能够用系统方法描述网络，并通过还原论实验测试其含义。我相信，这有力地证明了拟议工作的潜在影响，以及它的前景，使受训者在发现研究中具备出色和多样化的技能。

UNDERSTANDING RIBOSOME BIOGENESISThe overall objective of my research program is to understand how bacteria grow and survive. In the proposed research we will focus on the most basic of questions, to understand how the ribosome is assembled. The Escherichia coli ribosome is the product of assembly of a 16S ribosomal RNA and 21 ribosomal proteins for the 30S subunit, as well as a 5S rRNA, a 23S rRNA, and 34 proteins for the 50S subunit. Vintage studies by Nomura’s laboratory more than forty years ago revealed that functional ribosomal subunits could be assembled in a test tube with its component ribosomal RNAs and proteins. These and more contemporary studies have significantly advanced our understanding of the forces shaping subunit assembly. However, where these experiments have been performed under non-physiological conditions and times, it is noteworthy that E. coli assembles a ribosome extremely rapidly – in about two minutes – and it does so with more than 50 non-ribosomal protein factors. It is difficult to study ribosome assembly in vivo because assembly intermediates do not accumulate in amounts that permit isolation. Nevertheless, factors assisting ribosomal assembly represent extraordinary probes of ribosome maturation. Mutations directed at these factors can disable ribosome biogenesis and make it possible to isolate and characterize the products of perturbation. This approach is facilitating challenging studies of subtle aspects of folding and assembly that are the focus of this proposal. Most interestingly, an emerging group of biogenesis proteins, so-called ‘enigmatic factors’, are GTPases and RNA-binding proteins that appear to have chaperone or checkpoint roles in the late steps of the assembly process. Much of our work to date has focused on the YjeQ protein and has revealed a role in late steps of 30S subunit assembly. Evidence is mounting that several biogenesis factors constitute a functional network and prevent premature entry of late assembly intermediates into the translating pool of 70S ribosomes. For the 30S subunit the focus is on a cast of enigmatic factors that currently include BipA, Era, RbfA, RimM, RimP and YjeQ. A limited genetic interaction study in my laboratory revealed that overexpression of Era, a GTPase with a putative role in 30S assembly, was a suppressor of the slow growth and ribosome defects of the yjeQ deletion strain. Similarly, loss of the enigmatic 30S assembly factor RimM led to enhancement of these phenotypes. In the research proposed herein, we will expand our efforts to chart a genome-wide map of the functional interactions of these enigmatic factors in addition to exploring the implications with further studies of the assembly of the 30S ribosomal subunit. Our short-term goals are as follows:Objective 1. Probe the functional network of enigmatic factors with roles in 30S biogenesis.Objective 2. Test hypotheses posed by the functional network of enigmatic factors.Objective 3. Determine the nature of subunits that accumulate on depletion of biogenesis factorsWhere ribosome biogenesis is increasingly thought to proceed through multiple, parallel pathways guided by dispensable and redundant assembly factors, the proposed systems approach will be key to making impactful progress. My group’s unique capabilities in high throughput biology and conventional biochemistry will permit us to describe the network with systems approaches and to test the implications with reductionist experiments. I believe this argues strongly for the potential impact of the proposed work and its prospects to equip trainees with outstanding and varied skills in discovery research.