Interaction of Protein Factor e1F-4E with 5' mRNA Terminal Cap and Cap Analogs

这个项目是关于了解的机制， 真核细胞中mRNA翻译的起始。 通过 了解翻译的规律， 了解病毒感染细胞如何抑制正常蛋白质 合成和优先翻译病毒RNA以及如何生长 发展是有规律的。 这个项目的第一部分 包括描述起始因子的分子机制 eIF-4 E与5 ′ mRNA末端帽类似物的结合。 的结合 eIF-4 E将通过蛋白质荧光的变化来监测， 模拟荧光和圆二色性。 pH值的影响， 离子强度和温度将被检查。 从这些 数据，将确定是否存在优选的构象 iIF-4 E识别的帽的结构特征 对结合很重要的帽，以及 各种CAP衍生品。 在描述了这些初始反应之后， 结合所必需的mRNA的特征将通过使用 具有确定的结构差异的寡核苷酸。 的 其他起始因子和eIF-4 E磷酸化的影响 将对CAP识别进行测试。 这些互动将是 通过直接测定平衡常数来定量。 这样的研究将导致更精确的描述 翻译的启动机制。 该信息可以 适用于其他蛋白质-核酸相互作用。

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 translate viral RNAs and how growth and development are regulated. The first part of this project involves describing the molecular mechanism for initiation factor eIF-4E binding to 5' mRNA terminal cap analogs. The binding of eIF-4E will be monitored by changes in protein fluorescence, cap analog fluorescence, and circular dichroism. The effects of pH, ionic strength, and temperature will be examined. From these data it will be determined if there is a preferred conformation of the cap that is recognized by iIF-4E, the structural features of the cap important for binding,and the relative affinity of various cap derivatives. After characterizing these initial reactions, the structural features of mRNA necessary for binding will be examined by using oligonucleotides with defined structural differences. The effects of other initiation factors and phosphorylation of eIF-4E on cap recognition will be tested. These interactions will be quantitated by direct determination of equilbrium constants. Such studies will lead to a more precise description of the mechanism of initiation of translation. This information may be applicable to other protein-nucleic acid interactions.