The past few years have been highlighted by two major trends in eukaryotic protein syntheses. The first is the cloning of almost all of the polypeptide chains that constitute the initiation, elongation and termination factors. The second has been the development of the complexity with which the mRNA activation process is regulated as seen through the emergence of the eIF4E-binding proteins. The studies proposed in this application address three specific hypotheses. These hypotheses and the proposed experiments are outlined below: 1. eIF4A is an ATP-dependent RNA helicase and its action as a helicase is responsible for ATP-dependent scanning. This hypothesis will be tested by the following: characterization of the helicase activity of eIF4A in the presence of eIF4B, eIF4F and eIF4H; determination of the active sites for RNA binding in the mRNA-specific factors eIF4A, eIF4B, eIF4F and eIF4H; determination of the components necessary for scanning; testing of the mRNA specific factors for their effect on non-standard initiation events. 2. eIF4H and eIF4B are functional isoforms. This hypothesis will be tested by: preparing recombinant eIF4H and recombinant site-directed mutants of the RNA recognition motif (RRM) in eIF4H. eIF4H and eIF4B will be compared for their equivalent ability to participate in model assays and their influence on the selection of translational open reading frames. 3. eIF2A and eIF5B facilitates initiation at non-AUG codons. Both of these proteins participate in methionylpuromycin synthesis and polyphenylalanine synthesis at low Mg+2. We will test for their equivalence in influencing start site selection in a variety of non-standard mRNAs. The results of these studies should improve our understanding of the biochemical mechanism of RNA/RNA duplex unwinding in DEAD box RNA helicases and the initiation events associated with the binding of factors to mRNAs and their subsequent use in mRNA scanning. In addition, these experiments should also provide information on the mechanism of initiation of rare translation events(use of non-AUG start codons, internal initiation and re-initiation).
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