Studies of Ribosome Biogenesis

核糖体是负责在每个活细胞中合成蛋白质的RNA-蛋白质复合体。埃希里希菌 大肠杆菌核糖体，其分子量为2.5 mda，由两个亚基组成；小的(SSU)和 大亚基(LSU)。正确组装的LSU的三维结构从 结晶数据；然而，关于LSU组装的知识是有限的。这方面的知识对 促进针对核糖体组装的新型抗生素的设计。这项提议的目标是获得一个 对体内大肠杆菌LSU组装有详细的了解。LSU组装涉及RNA折叠、加工 和修饰，r-蛋白结合，以及成熟因子的结合和释放。DbpA，一个死胡同 RNA解旋酶是LSU成熟因子之一。解旋酶失活DbpA构建体的表达， R331a，产生三个LSU颗粒在细胞内的积累。随着时间的推移，这三个粒子会转化为50秒 因此，它们是LSU组装的中间体，而不是LSU组装的死胡同。此外， 这三种中间体分别属于LSU组装的三个不同阶段和三条平行的途径。因此， 他们的调查将提供有关不同LSU组装过程如何协调以及如何协调的信息 LSU组装的不同途径是相互连接的。据我们所知，这是唯一一个有三个 来自三条平行途径和LSU组装不同阶段的可分离中间体在细胞内积累； 因此，这是装配事件的协调和装配的互连的唯一系统 路径可以被调查。在目标1中，PI的实验室将确定rRNA的结构、修饰、 加工，以及三种LSU组装颗粒的r-蛋白质和成熟因子组成。比较 RRNA的结构、加工、修饰，以及r-蛋白质和成熟因子的组成 相互之间的中间体和50S亚基的高分辨晶体结构将识别：(I)rRNA LSU组装必须发生的结构异构化；(Ii)rRNA结构基序和r-蛋白在 RRNA转录后修饰和加工；(Iii)新的LSU成熟因子；(Iv)常见的 在所有LSU组装途径中错误折叠的rRNA基序。RRNA结构将通过化学方法进行探测 修改和下一代测序(NGS)。中间体的蛋白质组成将是 通过质谱学测定。在目标2中，PI的实验室将调查rRNA区域DbpA 在LSU组装过程中，催化核心直接作用于细胞内和颗粒中。紫外光交联剂 与NGS将用于确定DbpA催化核心区与rRNA的相互作用。RRNA区域 DbpA催化核心直接接触细胞内将告知：(I)LSU DbpA的多少区域起作用 以及(Ii)LSU组装的多少途径涉及DbpA。DbpA催化核的测定 颗粒中的天然底物与来自目标1和 正确组装的50S晶体结构数据将确定DbpA在LSU组装中的功能作用。

The ribosome is the RNA-protein complex responsible for protein synthesis in every living cell. The Escherichia coli (E. coli) ribosome, which has a molecular weight of 2.5 MDa, consists of two subunits; the small (SSU) and the large subunit (LSU). The three-dimensional structure of the properly assembled LSU is known from crystallographic data; however, the knowledge of LSU assembly is limited. This knowledge is invaluable to facilitate the design of novel antibiotics targeting ribosome assembly. The goal of this proposal is to gain a detailed understanding of in vivo E. coli LSU assembly. The LSU assembly involves RNA folding, processing and modification, r-proteins binding, and the association and release of maturation factors. DbpA, a DEAD-box RNA helicase, is one of the LSU maturation factors. Expression of the helicase inactive DbpA construct, R331A, produces the accumulation of three LSU particles in-cell. The three particles convert over time to 50S LSU; hence, they are LSU assembly intermediates and not dead-end products of LSU assembly. Moreover, the three intermediates belong to three different stages of LSU assembly and three parallel pathways. Hence, their investigation will produce information on how different LSU assembly processes are coordinated and how different pathways of LSU assembly are interconnected. To our knowledge, this is the only system where three isolatable intermediates from three parallel pathways and different stages of LSU assembly accumulate in-cell; thus, this is the only system in which the coordination of assembly events and the interconnectivity of assembly pathways can be investigated. In Aim 1, the PI's laboratory will determine the rRNA structure, modification, processing, and the r-protein and maturation factor compositions of three LSU assembly particles. Comparing the rRNA structure, processing, modifications, and the r-protein and maturation factor compositions of the intermediates to each other and to the high-resolution crystal structure of the 50S subunit will identify: (i) rRNA structural isomerizations that must occur for LSU assembly; (ii) rRNA structural motifs' and r-proteins' role in rRNA post-transcriptional modification and processing; (iii) novel LSU maturation factors; (iv) common misfolded rRNA motifs in all LSU assembly pathways. The rRNA structure will be probed by chemical modification and Next-Generation Sequencing (NGS). The protein composition of the intermediates will be determined by mass spectrometry. In Aim 2, the PI's laboratory will investigate the rRNA regions DbpA catalytic core directly acts upon during LSU assembly in-cell and in the particles. UV cross-linking combined with NGS will be used to determine DbpA catalytic core regions of interaction with rRNA. The rRNA regions that the DbpA catalytic core directly contacts in-cell will inform on: (i) how many regions of the LSU DbpA acts upon, and (ii) how many pathways of LSU assembly involve DbpA. Determination of the DbpA catalytic core's native substrates in the particles combined with the knowledge of the misfolded rRNA structure from Aim 1 and the properly assembled 50S crystal structure data will determine DbpA's functional role on LSU assembly.