Understanding ribosome biogenesis

了解核糖体生物遗传我研究计划的总体目标是了解细菌是如何生长和生存的。在拟议的研究中，我们将专注于最基本的问题，以了解核糖体是如何组装的。大肠杆菌核糖体是由30S亚基的16S核糖体RNA和21种核糖体蛋白质以及50S亚基的5S rRNA、23S rRNA和34种蛋白质组装而成的。40多年前野村实验室的陈年研究表明，功能性核糖体亚基可以与其组成部分核糖体RNA和蛋白质在试管中组装。这些和更多的当代研究极大地促进了我们对形成亚单位组装的力量的理解。然而，如果这些实验是在非生理条件和时间下进行的，值得注意的是，大肠杆菌极快地组装核糖体--大约两分钟--它利用50多个非核糖体蛋白因子做到这一点。在体内研究核糖体组装是困难的，因为组装中间体不会累积到允许分离的数量。然而，帮助核糖体组装的因素代表了核糖体成熟的特殊探针。针对这些因子的突变可以使核糖体生物发生失效，并使分离和鉴定扰动产物成为可能。这种方法有助于对折叠和组装的微妙方面进行具有挑战性的研究，这些方面是本提案的重点。最有趣的是，一组新出现的生物发生蛋白，即所谓的“谜团因子”，是GTP酶和RNA结合蛋白，它们似乎在组装过程的后期步骤中发挥伴侣或检查点的作用。到目前为止，我们的大部分工作都集中在YjeQ蛋白上，并揭示了在30s亚基组装的后期步骤中的作用。越来越多的证据表明，几种生物发生因子构成了一个功能网络，并防止晚期组装中间产物过早进入70年代核糖体的翻译池。对于30年代的亚基，重点是一系列神秘的因素，目前包括BIPA、Era、RbfA、Rimm、RimP和YjeQ。在我的实验室进行的有限的遗传交互作用研究表明，ERA的过度表达是yjeQ缺失菌株生长缓慢和核糖体缺陷的抑制因素。同样，神秘的30S组装因子Rimm的缺失导致了这些表型的增强。在这里提出的研究中，我们将进一步努力绘制这些神秘因子的功能相互作用的全基因组图谱，并探索30S核糖体亚基组装的进一步研究的意义。我们的短期目标如下：目标1.探索在30年代生物发生中起作用的神秘因子的功能网络。目标2.检验神秘因子功能网络提出的假说。目标3.确定生物发生因子耗尽时积累的亚基的性质。当人们越来越认为核糖体生物发生通过多条平行的途径进行时，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常规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，并用简化论实验测试其含义。我相信，这有力地证明了拟议工作的潜在影响及其使受训者掌握发现研究方面的杰出和各种技能的前景。

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.