Studies on start codon selection by eukaryotic ribosomes

描述（由申请人提供）：翻译起始异常通过不同机制导致恶性转化或其他慢性疾病。因此，对翻译起始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。在翻译起始过程中，真核起始因子1 （elF1）、elF5和elF2的所有三个亚基都与酵母mrna严格的AUG选择有关。elF5的c端结构域（CTD）结合elF1，并在elF2和5亚基因子elF3之间架起相互作用的桥梁，介导含有甲硫基引发剂tRNA的多因子复合物（MFC）的形成。当与核糖体结合时，MFC相互作用重排，允许elF5-CTD与elF4F复合物的elF4G亚基相互作用，并结合S'-cap mRNA，促进核糖体前起始复合物的形成。根据GM67841提案的先前目标，提供了证据表明翻译起始的准确性取决于MFC的功能，但该复合物如何组装和功能仍不清楚。特别是，将MFC和起始前复合物的功能与单个elf的结构联系起来是非常重要的，这已经开始揭示。在与哈佛医学院Gerhard Wagner博士的合作下，Asano小组已经解决了酵母elF1的结构，并确定了其与elF2和elFSc的NTDs以及elF4G和elF5的CTDs的界面。遗传和生化研究表明，在起始前复合物的组装和功能过程中，这些相互作用可能会重新排列。在这项更新提案中，第一个目标是与Wagner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诱变研究。第二个目标是从纯化的组分中重建完整的MFC，并利用它来了解其组装激活机制和elF5磷酸化的假设控制。最后，第三个目标是研究MFC在起始前复合体中的关键伙伴，elF4G-CTD和核糖体RNA。上述目的获得的数据将用于预测elF4G-CTD的elf5 - ctd结合面，并对后者进行定点诱变研究。在上述研究中获得的rRNA中的Ts-突变图谱将与在预测的40S亚基与mRNA的界面上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.