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

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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.