Mechanism of ribosomal subunit joining in eukaryotes

真核生物核糖体亚基连接机制

• 批准号: 6526026
• 负责人: TATYANA V PESTOVA
• 金额: $ 26.16万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6526026
• 关键词: chemical kinetics; cryoelectron microscopy; eukaryote; fluorescence spectrometry; gene mutation; guanosinetriphosphatases; hydrolysis; intermolecular interaction; messenger RNA; molecular assembly /self assembly; molecular genetics; protein localization; protein purification; protein reconstitution; protein structure function; ribosomal proteins; ribosomes; translation factor

DESCRIPTION (provided by applicant): Initiation of eukaryotic protein synthesis begins with separated 40S and 60S ribosomal subunits. The first step is formation of a 48S initiation complex at the initiation codon of mRNA. This process requires ternary complex, elFs 3, 1, 1A, 4A, 4B, 4F and a 40S subunit. The 40S subunit in 48S initiation complexes is associated with initiation factors and therefore can not immediately bind a 60S subunit. Joining of a 60S subunit to a 48S complex is associated with two linked events: hydrolysis of GTP bound to elF2 in the 48S complex and displacement of factors. Hydrolysis of elF2-bound GTP is stimulated by elF5. Recently we showed that hydrolysis of elF2-bound GTP is not sufficient to promote ribosomal joining as had previously been proposed, and that a second novel factor termed eIF5B is required. eIF5B is a homolog of the prokaryotic initiation factor IF2 and has a ribosome-dependent GTPase activity that is essential for its function. We shall identify and characterize structural elements of eIF5B responsible for the interaction of this factor with other translation components (40S and 60S subunits, Met-tRNAi, initiation factors) and the role of such interactions in subunit joining. Experiments will be done to determine whether eIF5 and eIF5B simply prepare 48S complexes for subsequent subunit joining or actively participate in the process. The roles of eIF5 and eIF5B in the displacement of initiation factors and the mechanism of displacement will also be determined. We will investigate the mechanism of ribosomal stimulation of eIF5B's GTPase activity and particular the role of ribosomal P proteins in this process. The localization of eIF5B on the ribosome will be studied by directed hydroxyl radical probing, chemical and enzymatic foot-printing and cryo-electron microscopy. These data will provide the basis for understanding the role of elF5B in subunit joining, and the mechanism of stimulation of eIF5B's GTPase activity by the ribosome. Fast kinetics techniques (fluorescence stopped-flow and quench-flow) will be applied to resolve individual steps and to study kinetic mechanism of two hydrolysis steps in subunit joining: the hydrolysis of elF2-bound GTP induced by elF5 and the hydrolysis of another GTP by elF5B.

描述(申请人提供)：启动真核蛋白质合成 以分离的40s和60s核糖体亚基开始。第一步是 在信使核糖核酸起始密码子上形成48S起始复合体。这 过程需要三元络合物，ELF 3，1，1A，4A，4B，4F和一个40S亚基。 48S起始复合体中的40S亚基与起始有关 因此而不能立即结合60S亚基。加入60年代 48S复合体的亚基与两个相连的事件有关： GTP结合elF2中的48S复合体和置换因子。水解性 ELF5刺激ElF2结合的GTP。最近我们证明了水解酶 ElF2结合的GTP不足以像以前那样促进核糖体连接 已经提出，并且需要第二个称为eIF5B的新因子。EIF5B 是原核生物起始因子IF2的同源物，具有 核糖体依赖的GTPase活性是其功能所必需的。 我们将确定和表征eIF5B的结构元素，该结构元素负责 这一因素与其他翻译成分(40秒和60秒)的相互作用 亚单位、Met-tRNAi、启动因子)以及这些相互作用在 亚基连接。将进行实验以确定eIF5和eIF5B 简单地为后续的亚基连接或主动地准备48S复合体 参与到这个过程中来。EIF5和eIF5B在骨肉瘤移位中的作用 还将确定引发因素和驱替机制。 我们将研究核糖体刺激eIF5B的GTP酶的机制 特别是核糖体P蛋白在这一过程中的作用。这个 EIF5B在核糖体上的定位将通过直接羟基进行研究 自由基探测、化学和酶足迹和冷冻电子 显微镜。这些数据将为理解 亚基连接中的elF5B及eIF5B的GTP酶激活机制 核糖体的活性。快速动力学技术(荧光停流 和急冷流)将被应用于解决个别步骤和研究 亚基连接中两步水解的动力学机制：水解酶 ELF5诱导的elF2结合的GTP和elF5B对另一种GTP的水解。