Characterization of 16S rRNA Maturation in E. coli

 描述（由申请人提供）：核糖体是一种复杂的核糖核蛋白（RNP），负责所有生物体中蛋白质合成的基本过程。核糖体的组装涉及核糖体RNA（rRNA）的转录和加工、核糖体蛋白的结合以及许多组装因子的活性。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。rRNA成熟的改变，从而与相关的核酸酶，这是参与这一过程，已被牵连作为致病性非常重要。对这种生物发生级联的清楚理解将是开发新型抗菌药物所必需的。然而，关于rRNA成熟的机制以及这一过程如何准确地融入核糖体生物发生级联反应仍然存在许多问题。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，从大肠杆菌中分离出前SSU组装中间体。杆菌从野生型E.大肠杆菌含有前体17 S rRNA，其在与细胞提取物或纯化的rRNA加工酶孵育后被加工成成熟的16 S rRNA。因此，这些数据表明，纯化的RNP是可以在体外经历成熟的途径上组装中间体。因此，本研究试图确定rRNA加工是如何整合到小核糖体亚基（SSU）生物发生中的，并破译SSU 16 S rRNA成熟的完整机制。coli中进行亲和纯化。目的1是从突变体E中分离和鉴定前SSU中间体。大肠杆菌菌株干扰SSU生物合成，其中不同的rRNA加工中间体积累。这些中间体的进一步表征将揭示作为rRNA加工底物的体内形成的RNP的组成，并揭示rRNA切割下游发生的关键组装事件。其次，利用这些纯化的pre-SSU作为底物，本提案的目标2将集中于使用细胞提取物和纯化的rRNA加工酶来动力学表征16 S rRNA体外成熟的分子机制。这些数据将作为理解各种条件下rRNA加工途径的通量的基础，并允许rRNA成熟酶的个体作用被解剖。综合这些目标，试图提供一个前所未有的一瞥16 S rRNA成熟和SSU的生物合成在E。杆菌

 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.