MUTATION OF THE CYSTEINE RESIDUES IN HUMAN INITIATION-FACTOR 4E - EFFECTS ON MESSENGER-RNA CAP-BINDING ABILITY

To investigate the role of four Cys residues of eukaryotic initiation factor (eIF)-4E in the recognition of the mRNA cap structure, single, double and quadruple mutant genes which encoded the Ala residues in th e place of the respective Cys residues were prepared using a synthetic human eIF-4E gene by the site-directed mutagenesis and were expressed in E. coil with the same way as the wild type. The cap binding abilit ies of respective mutated eIF-4Es were compared with that of the wild- type by m(7)GTP affinity chromatography. The results suggest that. alt hough all four of Cys residues participate in Be recognition or the mR NA cap structure, they contribute indirectly to the stabilization of o verall tertiary structure, especially of the cap binding pocket rather than by direct interaction. A comparison among the cap binding abilit ies of single, double and quadruple mutants indicated no existence of internal disulfide bonds in eIF-4E. (C) 1996 Academic Press, Inc.