Understanding ribosome biogenesis

了解核糖体生物发生 ** 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。 ** 大肠杆菌核糖体是一个16 S核糖体RNA和21个核糖体蛋白（30 S亚基）以及一个5S rRNA、一个23 S rRNA和34个蛋白（50 S亚基）组装的产物。 野村实验室40多年前的研究显示，功能性核糖体亚基可以在试管中与其组成核糖体RNA和蛋白质组装在一起。 这些和更多的当代研究显着推进了我们的理解力塑造亚基组装。 然而，当这些实验在非生理条件和时间下进行时，值得注意的是E.大肠杆菌组装核糖体的速度极快--大约在两分钟内--而且它能用50多种非核糖体蛋白因子来完成。 ** 在体内研究核糖体组装是困难的，因为组装中间体的积累量不允许分离。 然而，辅助核糖体组装的因子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生物合成失效，并使分离和表征扰动产物成为可能。 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“神秘因子”，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30 S亚基组装后期步骤中的作用。越来越多的证据表明，几个生源因素构成了一个功能网络，并防止过早进入后期组装中间体翻译池的70 S核糖体。 对于30 S亚基，重点是一系列神秘的因子，目前包括BipA，Era，RbfA，RimM，RimP和YjeQ。 在我的实验室有限的遗传相互作用的研究表明，Era的过度表达，一个假定的作用，在30 S组装的GTdR，是一个抑制剂的生长缓慢和核糖体缺陷的yjeQ缺失菌株。 类似地，神秘的30 S组装因子RimM的丢失导致这些表型的增强。 在本文提出的研究中，我们将扩大我们的努力，除了探索30 S核糖体亚基组装的进一步研究的影响之外，还将绘制这些神秘因子的功能相互作用的全基因组图谱。 我们的短期目标如下：** 目标1。 探索30 S生物发生中神秘因子的功能网络。目标2. 测试神秘因素的功能网络所提出的假设。目标3. 确定在生物合成因子耗尽时积累的亚基的性质 ** 在核糖体生物合成越来越多地被认为是通过多个平行途径进行的情况下，由重复和冗余组装因子指导，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，并用还原实验测试其影响。 我相信这有力地证明了拟议工作的潜在影响及其为学员提供发现研究方面的杰出和多样化技能的前景。

UNDERSTANDING RIBOSOME BIOGENESIS**The 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 factors**Where 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.