Studies of Ribosome Biogenesis

核糖体是RNA-蛋白质复合物，负责每个活细胞中的蛋白质合成。大肠 coli（E.大肠杆菌）核糖体，其分子量为2.5MDa，由两个亚基组成：小（SSU）和 大亚基（LSU）适当组装的LSU的三维结构由以下已知： 晶体学数据;然而，LSU组装的知识是有限的。这些知识是无价的， 有助于设计靶向核糖体组装的新型抗生素。该提案的目标是获得 详细了解体内E. coli LSU组装体。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，死亡盒 RNA解旋酶是LSU成熟因子之一。解旋酶失活DbpA构建体的表达， R331 A在细胞内产生三个LSU颗粒的积累。这三种粒子随着时间的推移转化为50 S LSU;因此，它们是LSU组装中间体，而不是LSU组装的死端产品。此外，委员会认为， 这三种中间体属于LSU组装的三个不同阶段和三条平行途径。因此，我们认为， 他们的调查将产生关于不同的LSU组装过程如何协调以及如何协调的信息。 LSU组装的不同途径相互连接。据我们所知，这是唯一的系统，其中三个 来自三个平行途径和LSU组装不同阶段的可分离中间体在细胞内积累; 因此，这是唯一的系统，其中装配事件的协调和装配的互连性 路径可以研究。在目标1中，PI的实验室将确定rRNA的结构，修饰， 加工，以及三个LSU组装颗粒的r-蛋白和成熟因子组成。比较 rRNA的结构、加工、修饰，以及R-蛋白和成熟因子的组成， 中间体相互之间以及与50 S亚基的高分辨率晶体结构之间的相互作用将鉴定：（i）rRNA 结构异构化，必须发生LSU组装;（ii）rRNA结构基序和r-蛋白的作用， rRNA转录后修饰和加工;（iii）新的LSU成熟因子;（iv）常见的 错误折叠的rRNA基序在所有LSU组装途径。rRNA结构将通过化学方法探测 修饰和下一代测序（NGS）。中间体的蛋白质组成将是 通过质谱法测定。在目标2中，PI的实验室将研究rRNA区域DbpA 催化核在电池内和颗粒中的LSU组装期间直接起作用。UV交联组合 将使用NGS测定DbpA与rRNA相互作用的催化核心区域。rRNA区域 DbpA催化核心直接接触电池内将告知：（i）LSU DbpA的多少区域起作用 上，以及（ii）有多少途径的LSU大会涉及DbpA。DbpA催化核的测定 颗粒中的天然底物结合来自Aim 1的错误折叠rRNA结构的知识， 正确组装的50 S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.