Studies on start codon selection by eukaryotic ribosomes

描述(申请人提供)：翻译启蒙是 蛋白质生物合成的限速步骤和一个重要的目标 基因表达的控制。不受监管的翻译启动可能会导致 细胞的恶性转化。酿酒酵母是一种 基因上易驯化的真核微生物，已被证明是一种 优秀的模型系统，用于研究各种细胞过程，包括 翻译启蒙。本项目的目的是了解 分子水平，以酿酒酵母为模式生物，如何真核 核糖体起始复合体严格选择AUG作为起始密码子。 由Donahue小组在 印第安纳大学涉及真核细胞启动因子1(ELFI)、ELF5和 在酵母mRNAs的严格8月选择中，elF2的所有三个亚基。ELF5 在正确的AUG识别时，作为elF2的GTP酶激活蛋白(GAP)。 最近，我在美国国立卫生研究院与辛尼布施博士合作，证明了一个由五个亚基组成的 因子elF3与eIF1和ELF5结合，C-末端结构域(CTD) ELF5在elF2和elF3之间的相互作用之间架起了桥梁。Elf1/elF3/ELF5/elF2 多因子复合体不含核糖体，含有化学计量比 甲硫基引发剂tRNA的量。当与核糖体结合时， 多因子复合体与elF4F结合(也通过ELF5-CTD)相互作用 M7G封端的mRNA.在这里，我假设，由 EIF5-CTD刺激形成40S核糖体预起始复合体和 也促进对AUG密码子的扫描和严格选择。我还提出了一个 ElF2 GTP酶如何在正确的AUG识别中被激活的模型 直接elF2-ELF5(GAP)相互作用。我想用以下工具测试这些型号 ELF5和elF4G的诱变研究 生化技术改编自对哺乳动物系统的研究。

DESCRIPTION (provided by applicant): Translation initiation is the rate-limiting step of protein biosynthesis and an important target for the control of gene expression. Unregulated translation initiation can result in malignant transformation of cells. The yeast Saccharomyces cerevisiae is a genetically tractable eukaryotic microorganism, which has proven to be an excellent model system to study a variety of cellular processes including translation initiation. The aim of this project is to understand at the molecular level, using S. cerevisiae as a model organism, how eukaryotic ribosomal initiation complexes stringently select AUG as a start codon. Extensive genetic and biochemical studies conducted by the Donahue group at the Indiana University implicated eukaryotic initiation factor 1 (elFi), elF5, and all three subunits of elF2 in the stringent AUG selection for yeast mRNAs. elF5 acts as a GTPase activating protein (GAP) for elF2 on correct AUG recognition. Working with Dr. HInnebusch at the NIH, I recently showed that a five-subunit factor elF3 binds to both eIF1 and elF5, and that the C-terminal domain (CTD) of elF5 bridges interaction between elF2 and elF3. The elF1/elF3/elF5/ elF2 multifactor complex occurs free of the ribosome and contains stoichiometric amount of the methionyl initiator tRNA. When bound to the ribosome, the multifactor complex interacts (also via elF5-CTD) with elF4F bound to m7G-capped mRNA. Here I hypothesize that the multiple interactions mediated by the eIF5-CTD stimulate formation of 40S ribosomal preinitiation complexes and also promote scanning and stringent selection of AUG codons. I also propose a model for how the elF2 GTPase is activated on correct AUG recognition via direct elF2-elF5 (GAP) interation. I would like to test these models with mutagenesis studies on elF5 and elF4G, in combination with sophisticated biochemical techniques adapted from studies on mammalian systems.