Studies from this laboratory and others from the last five years indicate that rhree distinct proteins are required specifically to bind mRNA to 43S preinitiation complexes and these three proteins are eukaryotic initiation factor (eIF) 4A, 4B and 4F. In the most general terms, eIF-4F recognizes the 5' m7G cap structure of eukaryotic mKNAs, eIF-4A is an ATP-dependent, single-stranded RNA binding protein, and eIF-4B coordinates their interaction. Our current efforts are designed to examine how these three factors (and perhaps others) are involved in the following: 1) How do 4OS subunits bind to mRNAs and move the mRNA to the initiating AUG codon? 2) Is the general mechanism for reinitiation (the downstream initiation of a bicistronic mRNA) the same as for the first initiation event or is it dissimilar? 3) Does internal initiation occur in polycistronic mRNAs (usually viral) and if so, by what mechanism? To examine these questions, we intend to use purified factors in both a fractionated system and a nuclease treated lysate and with SP6 transcripts to yield a series of """"""""mutated"""""""" mRNAs. These studies will extend our current knowledge as well as confirm previous notions on the function of the three mRNA specific initiation factors. In particular, these studies should offer unique insights into the initiation of uncapped mRNAs and polycistronic mRNAs, both usually only found in virally infected cells.
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