REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS INITITATION

真核蛋白质合成起始的调控

• 批准号: 2634607
• 负责人: ROBERT E. RHOADS
• 金额: $ 29.47万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-04-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2634607
• 关键词: Caenorhabditis elegans; genetic library; genetic promoter element; genetic regulation; intermolecular interaction; laboratory rabbit; messenger RNA; molecular cloning; oligonucleotides; protein biosynthesis; reporter genes; ribosomes; tissue /cell culture; translation factor

Protein synthesis plays a central role in growth and differentiation, response to change, and overall energy utilization of the cell. Viruses utilize the host translational machinery, and host cells have evolved complex antiviral responses. Deregulation of protein synthesis leads to malignant transformation. This project seeks to understand the rate- limiting step of protein synthesis, recruitment of mRNA to the ribosome, where much of translational control occurs. Two key factors in this process are eIF4E, the mRNA cap-binding protein, and eIF4G, a protein which links together the 40S ribosome, eIF4E, and the RNA helicase eIF4A. The roles of the eIF4 initiation factors will be studied by asking the following questions: 1. How do the eIF4 factors participate in recruitment of mRNA to the ribosome? We will arrest of intermediates of initiation complex assembly using allyloligonucleotides, overexpress eIF4G and a protease- resistant variant in cultured cells to determine oncogenic phenotype, test the effect of specific functional domains of eIF4G, and characterize eIF4E from C. elegans with respect to binding to m7G- and m3(2,2,7) G-containing caps. How does the phosphorylation of eIF4E affect protein synthesis? We will express in CREF cells eIF4E variants around the phosphorylation site to determine phenotype, examine the characteristics of eIF4E phosphorylation by PKCzeta, determine the properties of phosphorylated eIF4E by biophysical methods, and test whether eIF4E participates in a phosphorylation cycle during initiation. What physiological factors determine the intracellular levels and activity of eIF4G? We will study eIF4G synthesis, degradation and steady-state levels as a function several parameters as well as temperature-dependent conformational changes in recombinant eIF4G by biophysical and biochemical. How does the IRES of eIF4G mRNA direct internal initiation? We will delineate the borders of the eIF4G IRES, determine its secondary structure, characterize its translation in vivo, and attempt to find conditions for its utilization in vitro. And finally, what is the structure of the eIF4E gene? We will complete determination of the gene structure, define the promoter, and attempt to identify cis-acting regulatory elements.

蛋白质合成在生长和分化中起着核心作用， 对变化的反应，以及电池的整体能量利用率。病毒 利用宿主翻译机制，宿主细胞已经进化 复杂的抗病毒反应。放松对蛋白质合成的管制导致 恶变。这个项目试图了解- 限制蛋白质合成的步骤，向核糖体招募信使核糖体， 大部分的平移控制都发生在那里。这一过程中的两个关键因素 分别是eIF4E和eIF4G，eIF4E是一种mRNA帽结合蛋白，eIF4G是一种连接 将40S核糖体、eIF4E和RNA解旋酶eIF4A结合起来。的角色 EIF4的启动因素将通过以下问题进行研究 问题：1.eIF4因子如何参与mRNA的募集 核糖体吗？我们将逮捕起爆复合体的中间体 使用烯丙基寡核苷酸组装，过表达eIF4G和一种蛋白酶- 用培养细胞中的耐药变异体确定致癌表型 EIF4G特定功能结构域的作用及eIF4E特征 线虫与m7G-和m3(2，2，7)G-的结合 帽子。EIF4E的磷酸化如何影响蛋白质的合成？我们 将在CREF细胞中表达磷酸化位点周围的eIF4E变异体 要确定表型，请检查eIF4E的特征 用蛋白酪氨酸酶进行磷酸化，测定磷酸化产物的性质 EIF4E，并测试eIF4E是否参与了 启动过程中的磷酸化循环。哪些生理因素 测定细胞内eIF4G的水平和活性？我们会研究 EIF4G合成、降解和稳态水平作为几个函数 参数以及随温度变化的构象变化 通过生物物理和生化方法重组eIF4G。美国的IRES是如何 EIF4G信使核糖核酸直接内启动？我们将划定 EIF4G IRES，确定其二级结构，表征其翻译 在体内，并试图寻找体外利用的条件。和 最后，eIF4E基因的结构是什么？我们将完成 确定基因结构，定义启动子，并尝试 识别顺式作用的监管要素。