Mechanism of ribosomal subunit joining in eukaryotes

真核生物核糖体亚基连接机制

基本信息

批准号：
6917834
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
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-08-01 至 2007-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6917834
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

项目摘要

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（由申请人提供）：真核蛋白质合成的起始从分离的 40S 和 60S 核糖体亚基开始。第一步是在mRNA的起始密码子处形成48S起始复合物。这该过程需要三元复合物，ELF 3、1、1A、4A、4B、4F 和 40S 亚基。 48S起始复合物中的40S亚基与起始相关因此不能立即结合 60S 亚基。 60S的加入 48S 复合物的亚基与两个相关事件相关： GTP 与 48S 复合物中的 eF2 结合以及因子的置换。水解 eF5 刺激 eF2 结合的 GTP。最近我们发现水解 eF2 结合的 GTP 不足以像之前那样促进核糖体连接已被提出，并且需要第二个称为 eIF5B 的新因子。 eIF5B 是原核起始因子 IF2 的同源物，具有核糖体依赖性 GTP 酶活性对其功能至关重要。我们将识别和表征 eIF5B 的结构元素，负责该因子与其他翻译组件（40S 和 60S 亚基、Met-tRNAi、起始因子）以及此类相互作用在亚基连接。将进行实验以确定 eIF5 和 eIF5B 是否只需准备 48S 复合物以用于后续亚基连接或主动参与这个过程。 eIF5 和 eIF5B 在置换中的作用引发因素和位移机制也将被确定。我们将研究核糖体刺激eIF5B的GTPase的机制活性，特别是核糖体 P 蛋白在此过程中的作用。这 eIF5B 在核糖体上的定位将通过定向羟基研究自由基探测、化学和酶足迹以及低温电子显微镜。这些数据将为理解其作用提供基础 eIF5B在亚基连接中的作用以及eIF5B GTPase的刺激机制核糖体的活性。快速动力学技术（荧光停流和淬火流）将用于解决各个步骤并研究亚基连接中两个水解步骤的动力学机制：水解由elF5诱导的elF2结合的GTP以及由elF5B诱导的另一个GTP的水解。