Studies on start codon selection by eukaryotic ribosomes

调节异常的细胞因子起始通过不同的途径引起恶性转化或其他慢性疾病。 机制等因此，研究翻译启动机制有助于增进人类健康 和幸福该提案的长期目标是了解导致 翻译的准确起始。在翻译起始中，真核起始因子1（eIF 1）、eIF 5和所有 eIF 2的三个亚基与酵母mRNA的严格AUG选择有关。c-末端 eIF 5的结构域（CTD）结合eIF 1并桥接eIF 2和五亚基因子eIF 3之间的相互作用， 介导多因子复合物（MFC）的形成，所述多因子复合物还包含甲硫氨酰引发剂tRNA。当 结合到核糖体上，MFC相互作用重排，允许eIF 5-CTD与eIF 4G亚基相互作用 eIF 4F复合物与S '-加帽mRNA结合并促进核糖体预起始复合物的形成。 根据本GM 67841提案中的先前目标，提供了证据证明翻译的准确性 启动取决于MFC的功能，但这种复合物如何组装和发挥作用仍不清楚。 特别是，将MFC和预引发复合物的功能与细胞的功能相关联是非常重要的。 个人elf的结构，这已经开始揭开面纱。与哈佛的格哈德瓦格纳博士合作 医学院，浅野组已经解决了酵母elF 1的结构，并确定了其接口， eIF 2和eIF 5的NTD以及eIF 4G和eIF 5的CTD。遗传和生物化学研究表明 这些相互作用可能在前启动复合体组装和功能期间重新排列。在这次更新中， 在该提案中，第一个目标是与瓦格纳小组合作解决eIF 5-CTD的结构，并使用 这一信息指导其精心设计的诱变研究。第二个目标是重建完整的MFC 从纯化的成分，并用它来了解其组装激活的机制， 假设由eIF 5磷酸化控制。最后，研究了MFC的关键合作伙伴 在前起始复合物、eIF 4G-CTD和核糖体RNA中。从以前的目标中获得的数据 将用于预测eIF 4G-CTD的eIF 5-CTD结合面并进行定点诱变研究 关于后者。根据先前提案中的目标获得的rRNA中的>30个Ts-突变映射将是 与在预测的40 S亚基与mRNA的界面处引入的新突变一起使用。

Dysregulated translaion initiation causes malignant transformation or other chronic diseases by different mechanisms. Therefore, studies on the mechanism of translation initiation help to enhance human health and well being. The long-term goal of this proposal is to understand the complex pathway leading to the accurate initiation of translation. In translation initiation, eukaryotic initiation factor 1 (elF1), elF5, and all three subunits of elF2 are implicated in the stringent AUG selection for yeast mRNAs. The C-terminal domain (CTD) of elF5 binds elF1 and bridges interaction between elF2 and a five-subunit factor elF3, mediating fomration of the multifactor complex (MFC) which also contains the methionyl initiator tRNA. When bound to the ribosome, the MFC interactions rearrange, allowing elF5-CTD to interact with the elF4G subunit of elF4F complex bound to S'-capped mRNA and promoting formation of ribosomal preinitiation complex. Under the previous aims in this GM67841 proposal, evidence was provided that the accuracy of translation initiation depends on the function of the MFC, but how this complex assembles and functions is still unclear. In particular, it is very important to correlate the functions of the MFC and preinitiation complexes to the structure of individual elFs, which has started to unveil. In collaboration with Dr. Gerhard Wagner at Harvard Medical School, the Asano group has solved the structure of yeast elF1 and identified its interfaces to the NTDs of elF2¿ and elFSc and the CTDs of elF4G and elF5. Genetic and biochemical studies showed how these interactions may rearrange during preinitiation complex assembly and function. In this renewal proposal, the first aim is to solve the structure of elF5-CTD in collaboration with the Wagner group, and use this information to direct its well-designed mutagenesis studies. The second aim is to reconstitute full MFC from purified constituents, and use it to understand the mechanism of its assembly activation and hypothetical control by elF5 phosphorylation. Finally, the third aim is to study the critical partners of the MFC in the preinitiation complex, elF4G-CTD and the ribosomal RNA. The data obtained from the previous aims will be used to predict elF5-CTD-binding face of elF4G-CTD and perform site-directed mutagenesis studies on the latter. >30 Ts- mutations mapping in rRNA obtained under an aim in the previous proposal will be used together with new mutations to be introduced at the predicted 40S subunit interface to mRNA.