Translational activation of uncapped mRNAs by the central part of human eIF4G is 5' end-dependent.
Translation initiation factor (eIF) 4G represents a critical link between mRNAs and 40S ribosomal subunits during translation initiation. It interacts directly with the cap-binding protein eIF4E through its N-terminal part, and binds eIF3 and eIF4A through the central and C-terminal region. We expressed and purified recombinant variants of human eIF4G lacking the N-terminal domain as GST-fusion proteins, and studied their function in cell-free translation reactions. Both eIF4G lacking its N-terminal part (aa 486-1404) and the central part alone (aa 486-935) exert a dominant negative effect on the translation of capped mRNAs. 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 eIF4G-mediated translation. First, the C-terminal eIF4A binding site is dispensable for activation of uncapped mRNA translation. Second, translation of uncapped mRNA still requires 5' end-dependent ribosome binding. These new findings are incorporated into existing models of mammalian translation initiation.
De Gregorio
E
E
Gene Expression Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Preiss
T
T
Hentze
M W
MW
D004926:Escherichia coli / Q000235:genetics; D039603:Eukaryotic Initiation Factor-4G; D006801:Humans; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D016376:Oligonucleotides, Antisense; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000378:metabolism
D039603:Eukaryotic Initiation Factor-4G; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D016376:Oligonucleotides, Antisense; D010448:Peptide Initiation Factors; D012315:RNA Caps; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins