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）：真核蛋白合成的启动从分离的40和60s核糖体亚基开始。第一步是在mRNA的启动密码子上形成48S启动复合物。这过程需要三元复合体，小精灵3、1、1a，4a，4b，4f，4 40s亚基。 48S启动复合物中的40S亚基与启动有关因素，因此无法立即结合60S亚基。加入60年代 48S复合体的亚基与两个链接事件有关： GTP与48S复合物中的ELF2结合并取代因素。水解的 ELF5刺激ELF5刺激ELF2的GTP。最近我们表明了 ELF2结合的GTP不足以促进核糖体连接已提出，并且需要第二个称为EIF5B的新因素。 EIF5B 是原核生物起始因子IF2的同源物，并且具有核糖体依赖性GTPase活性对于其功能至关重要。我们将确定并表征负责EIF5B的结构元素该因子与其他翻译组件的相互作用（40s和60s 亚基，met-trona，起始因子）以及这种相互作用在亚基加入。将进行实验以确定EIF5和EIF5B是否只需准备48S复合体以进行后续亚基或积极加入或积极参加该过程。 EIF5和EIF5B在位移中的作用起始因子和位移机制也将得到确定。我们将研究EIF5B GTPase的核糖体刺激的机制核糖体P蛋白在此过程中的活性和特定的作用。这核糖体上EIF5B的定位将通过定向羟基研究根部探测，化学和酶脚印以及冷冻电子显微镜。这些数据将为理解角色的作用提供基础亚基连接中的ELF5B以及刺激EIF5B GTPase的机制核糖体的活性。快速动力学技术（荧光停止流量将应用Quench-Flow）来解决单个步骤并研究亚基连接的两个水解步骤的动力学机制： ELF5诱导的ELF2结合的GTP和ELF5B对另一GTP的水解。