INTERACTION OF EUKARYOTIC INITIATION FACTORS WITH MRNA

这个项目关注的是了解启动的机制 mRNA在真核细胞中的翻译。 通过了解法规 我们将更好地了解病毒感染的细胞如何抑制 正常的蛋白质合成和优先翻译的病毒RNA，以及如何 生长和发育受到调控。 项目的第一部分 描述了用于解旋酶活性的荧光测定法的发展 真核起始因子eIF-4A、eIF-4F和eIF-4 B。 这些测定 将允许动态事件被监测。 这些试验将允许 确定RNA解旋的动力学途径和解旋速率 在这个过程中的限制步骤。 蛋白质的化学计量 将确定解旋酶测定中的相互作用。 如果这些因素 其功能是结合单链RNA并防止再退火， 预期相对高的因子/核苷酸比率。 如果所述确定 第一步是最初的结合和解旋，一个低得多的因素 预计需要。 我们将描述因子RNA 各种结构不同的RNA相互作用， 盐和pH条件的范围。 我们将确定 互动。 这些结果将被量化并用于开发 解旋酶活性的更详细机制。

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.