IDENTIFICATION OF THE CAP-BINDING DOMAIN OF HUMAN RECOMBINANT EUKARYOTIC PROTEIN-SYNTHESIS INITIATION-FACTOR 4E USING A PHOTOAFFINITY ANALOG

Binding of eIF-4E to the 5' m(7)G cap structure of eukaryotic mRNA sig nals the initiation of protein synthesis. In order to investigate the molecular basis for this recognition, photoaffinity labeling with [gam ma-P-32]8-N(3)GTP was used in binding site studies of human recombinan t cap binding protein, (r)eIF-4E. Competitive inhibition of this cap a nalogue by m(7)GTP and capped mRNA indicated probe specificity for int eraction at the protein binding site. Saturation of the binding site w ith [gamma-P-32]8-N(3)GTP further demonstrated the selectivity of phot oinsertion. Aluminum (III)-chelate chromatography and reverse-phase HP LC were used to isolate the binding site peptide resulting from digest ion of photolabeled (r)eIF-4E with modified trypsin. Amino acid sequen cing identified the binding domain as the region containing the sequen ce Trp 113-Arg 122. Lys 119 was not identified in sequencing analysis nor was it cleaved by trypsin. These results indicate that Lys 119 is the residue directly modified by photoinsertion of [gamma-P-32]8-N(3)G TP. A detailed understanding of eIF-4E . m(7)G mRNA cap interactions m ay lead the way to regulating this essential protein-RNA interaction f or specific mRNA in vivo.