Assembling an understanding of ribosome biosynthesis

描述(申请人提供)：翻译的基本和必要过程是由核糖体催化的，本提案侧重于核糖体的生物发生。核糖体是一种复杂的RNPs，其生物发生涉及rRNA的转录、加工、修饰和折叠以及r-蛋白与至少40个组装因子的动态结合。长期以来，核糖体一直是了解复杂RNPs功能的基准，揭开30S亚单位生物发生机制的工作同样将增加我们对所有细胞RNPs生物发生的理解。我们的长期目标是详细了解大肠杆菌核糖体亚单位生物发生中涉及的因素和途径。由于细菌和真核核糖体的生物发生有几个共同的特征，这些研究结果无疑将导致对真核核糖体生物发生的新的保守特征和新的见解。此外，由于核糖体生物发生在细菌中可能是高度保守的，并与某些细菌的致病性有关，因此对细菌特异生物发生事件的发现将产生一些新的可能的抗微生物靶点，并可能对改变病原菌生长的医学操作方法产生新的见解。我们的方法包括分离体内形成的中间体，并随后分析这些颗粒的成分、结构和加工。初步结果显示存在三个不同的复合体，至少30个潜在的组装因子，其中三个我们已经确认为组装因子。我们将使用这些数据来进一步询问核糖体的生物发生，并确定这一过程中的重要步骤。我们将使用生物化学、遗传学、分子生物学和蛋白质组学来研究核糖体亚单位生物发生中间体和参与这些过程的因素，这些数据将被用来建立一个对细菌的这一过程至关重要的相互作用网络。这些研究不仅将为抗生素筛选和洞察带来新的目标 细菌耐药性的研究，但也将使我们更好地了解核糖体生物发生的演变，以及生物发生与细胞生理学基本方面的相互作用。

DESCRIPTION (provided by applicant): The fundamental and essential process of translation is catalyzed by the ribosome, and this proposal focuses on biogenesis of the ribosome. Ribosomes are intricate RNPs, whose biogenesis involves the transcription, processing, modification and folding of rRNA and the dynamic binding of r-proteins and at least 40 assembly factors. Ribosomes have long been the benchmark for understanding functional capabilities of complex RNPs, and work to unravel the mechanisms of 30S subunit biogenesis will similarly increase our understanding of biogenesis of all cellular RNPs. Our long-term goal is to gain a detailed understanding of the factors and pathways involved in E. coli ribosomal subunit biogenesis. Since bacterial and eukaryotic ribosome biogenesis share several features, results from these studies will undoubtedly lead to new conserved features and new insights into biogenesis of eukaryotic ribosomes. Furthermore, since ribosome biogenesis is likely to be highly conserved among bacteria and is linked to pathogenicity of some bacteria, discoveries of bacterial- specific biogenesis events will yield a number of new possible antimicrobial targets and likely new insights into possible methodologies for medical manipulation to alter growth of pathogenic bacteria. Our approach involves the isolation of in vivo formed intermediates and subsequent analysis of the components, structures, and processing of these particles. Preliminary results have shown the existence of three distinct complexes, at least 30 potential assembly factors, three of which we have already confirmed as assembly factors. We will use these data to further interrogate ribosome biogenesis and to identify important steps in this process. We will use biochemistry, genetics, molecular biology and proteomics to study ribosomal subunit biogenesis intermediates and factors that are involved in these processes and these data will be used to develop a network of interactions important for this process in bacteria. These studies will not only result in novel targets for antibiotic screening and insights into bacterial drug resistance, but will also yield better understanding of the evolution of ribosome biogenesis, and the interplay of biogenesis with fundamental aspects of cell physiology.