Fluorescence studies on association of human translation initiation factoreIF4E with mRNA cap-analogues

Binding of a long series of mono- and dinucleotide analogues of the 7-methy lguanosine containing 5'-mRNA-cap to human protein translation initiation f actor eIF4E has been investigated by means of fluorescence. A new methodolo gical approach in gathering and analysis of the fluorescence data provided us with very accurate values of the association equilibrium constant K and normalized, maximal quenching of the protein fluorescence Delta F-max, duri ng titration of eIF4E by various cap-analogues. The results confirm partici pation of at least two conserved tryptophan residues of eIF4E in interactio n with 7-methylguanine, as has been described recently for murine eIF4E, co mplexed with 7-methyl-GDP in crystal (Marcotrigiano et al., 1997, Cell 89, 951), and for yeast eIF4E, complexed with the same ligand in solution (Mats uo et al., 1997, Nature Struct. Biol. 4, 717). On the other hand binding by eIF4E of unmethylated guanine nucleotides and N-2,N-2,7-trimrthylguanine c ontaining nucleotides differ substantially from the way of binding of the r egular mRNA-cap. Influence of the structural features of the cap-analogues, especially the type of the second nucleoside in the dinucleotide caps, on their association with eIF4E and biological activities in in vitro protein translation systems has been discussed in light of the known structures of the eIF4E-7-methyl-GDP complexes in crystal and solution.