INTERACTION OF EUKARYOTIC INITIATION FACTORS WITH MRNA

这个项目关注于理解启动的机制。 真核细胞中信使核糖核酸的翻译。通过了解规则 通过翻译，我们将更好地了解病毒感染细胞是如何抑制 蛋白质的正常合成和优先翻译的病毒RNA及其方式 生长和发育是受监管的。项目的第一部分 描述了一种测定解旋酶活性的荧光分析方法的发展。 真核启动因子eIF-4A、eIF-4F和eIF-4B。这些化验 将允许动态事件被监控。这些化验将允许 核糖核酸解旋的动力学路径和速率的测定 限制这一过程中的步骤。蛋白质的化学计量比 解旋酶分析中的相互作用将被确定。如果这些因素 结合单链RNA并防止再退火的功能，a 相对较高的因子/核苷酸比率是预期的。如果这一决定 步骤是最初的捆绑和解开，一个低得多的因素 要求是预期的。我们将对因子RNA进行表征 各种结构不同的RNA和在 盐的范围和pH条件。我们将确定以下内容的协作性 相互作用。这些结果将被量化并用于开发 更详细的解旋酶活性机制。

This project is concerned with understanding the mechanism of initiation of mRNA translation in eucaryotic cells. By understanding the regulation of translation we will better understand how viral infected cells inhibit normal protein synthesis and preferentially translated viral RNAs and how growth and development are regulated. The first part of the project describes the development of a fluorescence assay for helicase activity of eucaryotic initiation factors eIF-4A, eIF-4F and eIF-4B. These assays will allow kinetic events to be monitored. These assays will allow the determination of the kinetic pathways in RNA unwinding and the rate limiting steps in this process. The stoichiometry of the protein interaction in the helicase assay will be determined. If the factors function to bind single stranded RNA and prevent reannealing, a relatively high factor/nucleotide ration is expected. If the determining step is the initial binding and unwinding, a much lower factor requirement is expected. We will characterize the factor RNA interactions for a variety of structurally different RNA and under a range of salt and pH conditions. We will determine the cooperativity of the interactions. These results will be quantitated and used to develop a more detailed mechanism of helicase activity.