TRANSLATIONAL ACTIVATION OF UNCAPPED MESSENGER-RNAS BY THE CENTRAL PART OF HUMAN EIF4G IS 5'-END-DEPENDENT

Translation initiation factor (elF) 4G represents a critical link betw een mRNAs and 40S ribosomal subunits during translation initiation. It interacts directly with the cap-binding protein elF4E through its N-t erminal part, and binds elF3 and elF4A through the central and C-termi nal region. We expressed and purified recombinant variants of human el F4G lacking the N-terminal domain as GST-fusion proteins, and studied their function in cell-free translation reactions. Both elF4G lacking its N-terminal part (aa 486-1404) and the central part alone (aa 486-9 35) exert a dominant negative effect on the translation of capped mRNA s. Furthermore, these polypeptides potently stimulate the translation of uncapped mRNAs, Although this stimulation is cap-independent, it is shown to be dependent on the accessibility of the mRNA 5' end. These results reveal two unexpected features of elf4G-mediated translation. First, the C-terminal elF4A binding site is dispensable for activation of uncapped mRNA translation. Second, translation of uncapped mRNA st ill requires 5' end-dependent ribosome binding, These new findings are incorporated into existing models of mammalian translation initiation .