Studies on start codon selection by eukaryotic ribosomes

描述(申请人提供)：翻译启动失调可通过不同的机制导致恶性转化或其他慢性疾病。因此，对翻译启动机制的研究有助于增进人类的健康和福祉。这一建议的长期目标是了解导致准确启动翻译的复杂途径。在翻译起始中，真核细胞起始因子1(Elf1)、ELF5和elF2的所有三个亚基都参与了酵母mRNAs的严格AUG选择。ELF5的C-末端结构域(CTD)与ElF1结合，并在ElF2和一个五亚基因子elF3之间架起相互作用的桥梁，介导多因子复合体(MFC)的形成，该复合体还包含甲硫基启动子tRNA。当与核糖体结合时，MFC相互作用重排，允许ELF5-CTD与结合在S封顶的mRNA上的elF4F复合体的elF4G亚基相互作用，促进核糖体预起始复合体的形成。根据GM67841提案先前的目标，有证据表明，翻译启动的准确性取决于MFC的功能，但这个复合体是如何组装和发挥作用的仍不清楚。特别是，将MFC和预引发复合体的功能与单个ELF的结构联系起来是非常重要的，这一结构已经开始揭开面纱。与哈佛医学院的Gerhard Wagner博士合作，Asano团队已经解决了酵母Elf1的结构，并确定了它与elF2和elFSc的NTDS以及elF4G和ELF5的CTD的接口。遗传和生化研究表明，在预启动复合体组装和功能过程中，这些相互作用可能会重新排列。在这项更新建议中，第一个目标是与瓦格纳小组合作解决ELF5-CTD的结构，并利用这些信息指导其精心设计的突变研究。第二个目的是从纯化的成分中重建完整的MFC，并利用它来理解其组装激活和ELF5磷酸化的假说控制的机制。最后，第三个目的是研究MFC在预引发复合体中的关键伙伴elF4G-CTD和核糖体RNA。从前面的目标获得的数据将被用于预测elF4G-CTD的ELF5-CTD结合面，并对后者进行定点突变研究。根据先前提案中的目标获得的rRNA中的TS-突变映射将与将在预测的40S亚基与mRNA的交界处引入的新突变一起使用。

DESCRIPTION (provided by applicant): Dysregulated translation 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 formation 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.