Characterization of 16S rRNA Maturation in E. coli

 描述(申请人提供)：核糖体是一种复杂的核糖核蛋白(RNP)，负责所有生物体内蛋白质合成的基本过程。核糖体的组装涉及核糖体RNA(RRNAs)的转录和加工、核糖体蛋白的结合以及多种组装因子的活性。核糖体组装过程中的缺陷与各种人类病原体的毒力和耐药性的变化有关。参与这一过程的rRNA成熟和相关核酸酶的变化被认为对致病性非常重要。为了开发新的抗菌药物，需要清楚地了解这种生物发生级联反应。然而，关于rRNA成熟的机制以及这一过程到底如何适应核糖体生物发生级联仍然存在许多问题。为了解决这些问题，卡尔弗实验室开发了一种新的亲和纯化技术，从大肠杆菌中分离前SSU组装中间体。从野生型大肠杆菌中纯化的Pre-SSU含有一个前体17S rRNA，与细胞提取液或纯化的rRNA加工酶孵育后，被加工成成熟的16S rRNA。因此，这些数据表明，纯化的RNPs是可以在体外成熟的途径上组装的中间体。因此，本建议旨在确定rRNA处理如何整合到小核糖体亚单位(SSU)的生物发生中，并利用这种亲和纯化方法破译SSU 16S rRNA在大肠杆菌中成熟的完整机制。本建议的目的是从对SSU生物发生产生干扰的突变大肠杆菌菌株中分离和鉴定SSU前中间体，在该突变株中积累不同的rRNA加工中间体。对这些中间体的进一步表征将揭示体内形成的RNPs的组成，这些RNPs是rRNA加工的底物，并揭示发生在rRNA切割下游的关键组装事件。其次，以这些纯化的Pre-SSU为底物，本提案的目标2将重点放在使用细胞提取液和纯化的rRNA加工酶来研究16S rRNA体外成熟的分子机制。这些数据将为了解各种条件下rRNA加工途径的通量奠定基础，并允许解剖rRNA成熟酶的单个角色。综上所述，这些目的试图提供一个前所未有的一瞥16S rRNA成熟和SSU在大肠杆菌中的生物发生的机制。

 DESCRIPTION (provided by applicant): The ribosome is a complex ribonucleoprotein (RNP) responsible for the essential process of protein synthesis in all living organisms. Assembly of ribosomes involves the transcription and processing of ribosomal RNAs (rRNAs), association of ribosomal proteins, and the activities of many assembly factors. Defects in the ribosome assembly process are associated with changes in virulence and drug resistance in various human pathogens. Alterations in rRNA maturation and thus with associated nucleases, which are involved in this process, have been implicated as being very important for pathogenicity. A clear understanding of this biogenesis cascade will be needed for the development of novel antimicrobial drugs. However, many questions remain regarding the mechanism of rRNA maturation and exactly how this process fits into the ribosome biogenesis cascade. To address these questions the Culver laboratory has developed a novel affinity purification technique to isolate pre-SSU assembly intermediates from E. coli. Purified pre-SSUs from wild-type E. coli contain an precursor 17S rRNA that upon incubation with cell extracts or purified rRNA processing enzymes is processed into mature 16S rRNA. Thus these data suggest that purified RNPs are on- pathway assembly intermediates that can undergo maturation in vitro. Therefore, this proposal seeks to determine how rRNA processing is integrated into small ribosomal subunit (SSU) biogenesis and to decipher the complete mechanism of SSU 16S rRNA maturation in E. coli using this affinity purification method. Aim1 of this proposal is to isolate nd characterize pre-SSU intermediates from mutant E. coli strains perturbed for SSU biogenesis in which distinct rRNA processing intermediates accumulate. Further characterization of these intermediates will divulge the composition of in vivo formed RNPs that are the substrates for rRNA processing and reveal critical assembly events that occur downstream of rRNA cleavage. Secondly, using these purified pre-SSUs as substrates, Aim 2 of this proposal will focus on using cell extracts and purified rRNA processing enzymes to kinetically characterize the molecular mechanism of 16S rRNA maturation in vitro. These data will serve as a foundation for understanding the flux of rRNA processing pathways under various conditions and allow the individual roles of rRNA maturation enzymes to be dissected. Taken together these aims seek to provide an unprecedented glimpse into the mechanism of 16S rRNA maturation and SSU biogenesis in E. coli.