Studies on start codon selection by eukaryotic ribosomes

描述（由申请人提供）：翻译启动是 蛋白质生物合成的限速步骤和一个重要的目标， 控制基因表达。不受调控的翻译起始可导致 细胞的恶性转化。酿酒酵母是一种 遗传上易处理的真核微生物，已被证明是一种 优秀的模型系统，研究各种细胞过程，包括 翻译起始该项目的目的是了解 分子水平上，利用S.酿酒酵母作为模式生物，真核生物如何 核糖体起始复合物严格选择AUG作为起始密码子。 Donahue小组进行了广泛的遗传和生物化学研究， 印第安纳州大学的研究表明，真核起始因子1（eIF 1）、eIF 5和 eIF 2的所有三个亚基在酵母mRNA的严格AUG选择中。elF5 在正确的AUG识别上充当eIF 2的GTP酶激活蛋白（GAP）。 最近，我与美国国立卫生研究院的Hinnebusch博士合作， 因子eIF 3与eIF 1和eIF 5结合，C-末端结构域（CTD） eIF 5的结合是eIF 2和eIF 3之间相互作用的桥梁。elF1/elF3/elF5/ elF2 多因子复合物不含核糖体， 甲硫氨酰引发剂tRNA的量。当与核糖体结合时， 多因子复合物与结合至 m7 G加帽mRNA。在这里，我假设，多重相互作用介导的 eIF 5-CTD刺激40 S核糖体前起始复合物形成， 也促进AUG密码子的扫描和严格选择。我还提出了一个 关于eIF 2 GTdR如何在正确的AUG识别上被激活的模型， 直接eIF 2-eIF 5（GAP）相互作用。我想测试一下这些模型 对eIF 5和eIF 4G的诱变研究，与复杂的 从哺乳动物系统的研究中改编的生化技术。

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.