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

[9722907] Goss该项目的长期目标是了解真核蛋白合成的机制。真核生物在正常细胞中启动转译的方式与病毒感染细胞或在热休克等应激条件下启动转译的方式不同。蛋白质合成的总体方案已被描述，但细节，特别是动力学的这一过程仍有待阐明。真核起始因子（eIFs）与mRNA和寡核苷酸相互作用的平衡和动力学将被检查。eIFs对核糖体与几种寡核苷酸和mRNA结合的影响将被确定。直接荧光测量将用于阐明起始因子，核糖体和mRNA或寡核苷酸的相互作用。将分析这些多重相互作用，以确定结合相互作用是合作的、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途径。定量数据将有助于深入了解mRNA二级结构对核糖体结合的影响，RNA 5‘和3’端之间可能的通信，以及在高等植物系统中内部核糖体进入的可能性。这些结果将有助于形成真核起始复合物组装的详细分子机制。本项目将阐明植物系统中蛋白质合成的机制。蛋白质合成调控在细胞生长发育调控以及生物技术应用中具有重要意义。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，而不会产生用于基因克隆的细菌或其他宿主可能产生的污染物。因此，了解植物中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产生的相互作用的限速步骤。通过阐明这些步骤，我们可以确定可能的干预点，以增加或降低整个蛋白质合成过程的速率。

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.