Mutation of the cysteine residues in human initiation factor 4E: effects on mRNA 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 the 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. coli with the same way as the wild type. The cap binding abilities of respective mutated eIF-4Es were compared with that of the wild-type by m7GTP affinity chromatography. The results suggest that, although all four of Cys residues participate in the recognition of the mRNA cap structure, they contribute indirectly to the stabilization of overall tertiary structure, especially of the cap binding pocket, rather than by direct interaction. A comparison among the cap binding abilities of single, double and quadruple mutants indicated no existence of internal disulfide bonds in eIF-4E.