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

本项目致力于了解真核细胞中信使核糖核酸翻译的启动机制。通过了解翻译的调控，我们将更好地了解病毒感染细胞如何抑制正常的蛋白质合成，并优先翻译病毒RNA，以及生长和发育是如何受到调控的。本项目的第一部分涉及描述起始因子eIF-4E与5‘mRNA末端帽类似物结合的分子机制。EIF-4E的结合将通过蛋白质荧光、帽类荧光和圆二色谱的变化来监测。将考察pH值、离子强度和温度的影响。根据这些数据，将确定是否存在IIF-4E识别的帽子的优选构象、帽子的结构特征对结合很重要，以及各种帽子衍生物的相对亲和力。在确定了这些初始反应的特征后，将使用具有明确结构差异的寡核苷酸来检查结合所需的mRNA的结构特征。将测试其他启动因子和eIF-4E的磷酸化对帽识别的影响。这些相互作用将通过直接测定平衡常数来定量。这样的研究将有助于更准确地描述翻译的启动机制。这一信息可能适用于其他蛋白质-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.