Mechanism of ribosomal subunit joining in eukaryotes

描述（由申请人提供）：真核蛋白质合成的起始 开始于分离的40 S和60 S核糖体亚基。第一步是 在mRNA的起始密码子处形成48 S起始复合物。这 该过程需要三元复合物，eIF 3，1，1A，4A，4 B，4F和40 S亚基。 48 S起始复合物中的40 S亚基与起始有关， 因子，因此不能立即结合60 S亚基。加入60 s 48 S复合物的亚基与两个相关事件相关： GTP结合到48 S复合物中的eIF 2和因子的置换。水解 eIF 2结合的GTP被eIF 5刺激。最近，我们发现， eIF 2结合的GTP不足以促进核糖体连接，如先前所述。 已经提出，并需要称为eIF 5 B的第二个新因子。eIF5B 是原核起始因子IF 2的同源物， 核糖体依赖性GT3活性是其功能所必需的。 我们将确定和表征eIF 5 B的结构元件， 该因子与其他翻译成分（40 S和60 S）的相互作用 亚基，Met-tRNAi，起始因子）以及这种相互作用在 亚基连接。将进行实验以确定eIF 5和eIF 5 B是否 简单地制备48 S复合物用于随后的亚基连接或主动地 参与这个过程。eIF 5和eIF 5 B在细胞凋亡中的作用 还将确定引发因素和位移机制。 我们将研究核糖体刺激eIF 5 B的GTP酶的机制 活性，特别是核糖体P蛋白在这一过程中的作用。的 将通过定向羟基研究eIF 5 B在核糖体上的定位 自由基探测，化学和酶足迹和低温电子 显微镜这些数据将为理解 eIF 5 B在亚基连接中的作用及其刺激eIF 5 B GT3表达的机制 核糖体的活动。快速动力学技术（荧光停流 和淬火流）将被应用于解决个别步骤，并研究 亚基连接中两个水解步骤的动力学机制： eIF 5诱导的eIF 2结合的GTP和eIF 5 B对另一GTP的水解。

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.