Characterization of the Interactions of Eukaryotic Initiation Factors, Ribosomes, and mRNA

9722907戈斯这个项目的长期目标是了解真核蛋白质合成的机制。真核生物在正常细胞中启动翻译的方式不同于病毒感染的细胞或在热休克等应激条件下的细胞。蛋白质合成的总体方案已经描述，但细节，特别是这一过程的动力学仍有待阐明。本课程将探讨真核生物起始因子与信使核糖核酸和寡核苷酸相互作用的平衡和动力学。EIFS对核糖体与几种寡核苷酸和信使核糖核酸结合的影响将被确定。直接荧光测量将被用来阐明启动因子、核糖体和mRNA或寡核苷酸之间的相互作用。将分析这些多个交互作用以确定绑定交互作用是合作的、反合作的还是独立的。动力学结果将决定限速步骤和可能的相互作用路径。定量数据将有助于深入了解RNA二级结构对核糖体结合的影响，RNA 5‘端和3’端之间可能的通讯，以及高等植物系统内部核糖体进入的可能性。这些结果将有助于形成组装真核起始复合体的详细分子机制。这个项目将阐明植物系统中蛋白质合成的机制。蛋白质合成的调节在调节细胞的生长和发育以及在生物技术应用中都很重要。植物为生产疫苗和其他具有商业价值的蛋白质提供了可能的宿主，而不存在用于基因克隆的细菌或其他宿主可能存在的污染物。因此，了解植物蛋白质合成的调控是很重要的。这些研究将确定决定蛋白质生产的相互作用的限速步骤。通过阐明这些步骤，可以确定可能的干预点，以增加或减少整个蛋白质合成过程的速度。

9722907 Goss The long term goal of this project is to understand the mechanism of eukaryotic protein synthesis. Eukaryotes initiate translation differently in normal cells than in virus infected cells or cells under stress conditions such as heat shock. The overall scheme of protein synthesis has been described, but the details and particularly the kinetics of this process remain to be elucidated. The equilibrium and kinetics of the interaction of eukaryotic initiation factors (eIFs) with mRNA and oligonucleotides will be examined. The effects of eIFs on ribosome binding to several oligonucleotides and mRNA will be determined. Direct fluorescence measurements will be used to elucidate the interaction of initiation factors, ribosomes and mRNA or oligonucleotides. These multiple interactions will be analyzed to determine if the binding interactions are cooperative, anti-cooperative, or independent. Kinetic results will determine the rate limiting steps and the probable pathway of interactions. Quantitative data will give insight into the effect of mRNA secondary structure on ribosome binding, the possible communication between the 5' and 3' termini of RNA, and the possibility of internal ribosome entry in higher plant systems. These results will aid in formulating a detailed molecular mechanism for the assembly of the eukaryotic initiation complex. This project will elucidate the mechanism for protein synthesis in plant systems. Regulation of protein synthesis is important in regulation of cell growth and development as well as in biotechnology applications. Plants provide possible hosts for production of vaccines and other proteins of commercial interest without the contaminants that may be present from a bacterial or other host used for gene cloning. It is therefore important to understand the regulation of protein synthesis in plants. These studies will determine the rate limiting steps for interactions that determine the production of proteins. By elucidating these steps o ne can then determine the possible intervention points to increase or decrease the rate of the overall protein synthesis process.