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.
