EFFECT OF MESSENGER-RNA CAP STRUCTURE ON EIF-4E PHOSPHORYLATION AND CAP-BINDING ANALYSES USING SER209-MUTATED EIF-4ES

The in vitro phosphorylation of human recombinant eIF-4E by protein ki nase C was most effective in the absence of m(7)GTP, supporting a 'per formed complex model' as the mRNA binding step of initiation, i.e., eI F-4E first forms an initiation complex eIF-4F and is phosphorylated be fore interacting with mRNA. On the other hand, the comparison of m(7)G TP-binding ability of wild-type eIF-4E with those of four Ser209-mutat ed ones (S209A, S209D, S209E and S209K) showed that the addition of an ionic charge on Ser209 increases the cap affinity of eIF-4E by repress ing the release of the cap from the complex, not by increasing the com plex formation, suggesting the importance of a retractable ionic bridg e between Ser209 and Lys159 in controlling the cap binding by eIF-4E p hosphorylation. (C) 1998 Academic Press.