Studies on start codon selection by eukaryotic ribosomes

真核核糖体起始密码子选择的研究

• 批准号: 7887080
• 负责人: KATSURA ASANO
• 金额: $ 19.97万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-10 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7887080
• 关键词: Binding; Biochemical; C-terminal; Chronic Disease; Collaborations; Complex; Data; Elements; Eukaryotic Initiation Factor-1; Face; Free Ribosome; GTPase-Activating Proteins; Genetic; Goals; Health; Human; In Vitro; Individual; Initiator Codon; Initiator tRNA; Malignant - descriptor; Maps; Mediating; Messenger RNA; Mutagenesis; Mutation; N-terminal; Pathway interactions; Phosphorylation; RNA, Transfer, Met; Ribosomal RNA; Ribosomes; Role; Site-Directed Mutagenesis; Structure; Translation Initiation; Yeasts; design; mRNA capping; medical schools; reconstitution

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.

失调的翻译倡议会导致恶性转化或其他慢性疾病。 机制。因此，关于翻译计划机制的研究有助于增强人类健康 幸福。该提议的长期目标是了解导致的复杂途径 准确的翻译计划。在翻译计划中，真核倡议因子1（ELF1），ELF5和所有 在严格的酵母mRNA选择中，暗示ELF2的三个亚基。 C末端 ELF5的域（CTD）结合ELF1和桥梁在ELF2和五亚基因子ELF3之间的相互作用， 介导多因子络合物（MFC）的Fomration，该复合物还包含甲基激发剂tRNA。什么时候 绑定到核糖体，MFC相互作用重新排列，允许Elf5-CTD与Elf4g亚基相互作用 Elf4f复合物的结合与S'Capped mRNA结合并促进核糖体前启动复合物的形成。 根据该GM67841提案的先前目的，有证据表明翻译的准确性 起始取决于MFC的功能，但是这种复杂的组装和功能仍不清楚。 特别是，将MFC的功能和预上络合物的功能与 单个小精灵的结构，该结构已开始揭幕。与哈佛大学的Gerhard Wagner博士合作 医学院，Asano集团解决了酵母Elf1的结构，并确定了其界面 Elf2¿和Elfsc的NTDS以及Elf4g和Elf5的CTD。遗传和生化研究表明了如何 这些相互作用可能会在预启用复合物组装和功能期间重新排列。在此续约中 提案，第一个目的是与瓦格纳集团合作解决Elf5-CTD的结构，并使用 这些信息是为了指导其精心设计的诱变研究。第二个目的是重建完整的MFC 从纯化的宪法中，并使用它来了解其组装激活的机制和 ELF5磷酸化的假设控制。最后，第三个目标是研究MFC的关键伙伴 在预上络合物中，ELF4G-CTD和核糖体RNA。从以前的目标获得的数据 将用于预测ELF4G-CTD的ELF5-CTD结合面并进行定位的诱变研究 在后者。在先前提案中获得的rRNA中> 30 ts-突变映射将是 与新的突变一起用于预测的40S亚基界面与mRNA。