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

该项目的长期目标是了解真核生物蛋白质合成的机制。真核细胞在正常细胞和病毒感染细胞或在热休克等应激条件下启动翻译的方式不同。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。提出了真核起始因子与mRNA和寡核苷酸相互作用的平衡和动力学的详细研究。直接荧光测量将用于阐明起始因子和mRNA或寡核苷酸的相互作用。先前已经分析了与5'非编码区结合的几个起始因子的相互作用。这些相互作用的动力学现在将被确定。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。这些结果将有助于制定一个详细的分子机制，为真核起始玉米粒组装。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。荧光光谱学将用于测量这些相互作用，并确定这一过程的细节。通过阐明速率限制步骤，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。＊＊＊

Goss 9600521 The long term goal of this project is to understand the mechanism of eukaryotic protein synthesis. Eukaryotes initiate translation differently in normal cell and in viral infecled cell or cells under stress conditions such as heat shock. The overall scheme of protein synthesis has been described, but the details and particularily the kinetics of this process remain to be elucidated. A detailed study of the equilibrium and kinetics of the interaction of eukaryotic initiation factors with mRNA and oligonucleotides is proposed. Direct fluorescence measurements will be used to elucidate the interaction of initiation factors and mRNA or oligonucleotides. The interaction of several initiation factors that bind to the 5' noncoding region have been previously analyzed. The kinetics of these interactions will now be determined. 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. These results will aid in formulating a detailed molecular mechanism for the assembly of the eukaryotic initiation cornplex. %%% 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 vacines and other proteins of commercial interest without the contaminants that may be present from a bacterial or other host used for gene cloning. lt 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. Fluorescence spectroscopy will be used to measure these interactions and determine the details of this process. By elucidating the rate limiting steps one can then determine the possible intervention points t o increase or decrease the rate of the overall protein synthesis process. ***