Analysis of the mRNA Cap‐Binding Ability of Human Eukaryotic Initiation Factor‐4E by Use of Recombinant Wild‐Type and Mutant Forms

Abstract

In order to identify the amino acid residues necessary for the selective recognition of the mRNA cap structure by human eukaryotic initiation factor-4E (eIF-4E), which plays a central role in the first step of mRNA translation, we prepared recombinant wild-type and fourteen mutant forms and compared their cap-binding abilities by affinity chromatography. By the direct expression of a synthetic gene encoding human eIF-4E as the soluble form in Escherichia coli and the application on a 7-methylguanosine-5'-triphosphate-Sepharose 4B cap affinity column, pure recombinant eIF-4E was prepared; the optimum pH for the binding of the mRNA cap was 7.5. Among the amino acid residues conserved among various eIF-4E species, each of 14 functional residues was replaced with a nonpolar amino acid (alanine or leucine). All mutant eIF-4E genes, which were constructed by site-directed niutagenesis, were expressed in the same way as the wild type, and their cap-binding abilities were compared with that of the wild type. Consequently, all eight tryptophan residues, Glu103, and two histidine residues at positions 37 and 200 in human recombinant eIF-4E were suggested to be important for the recognition of the mRNA cap structure through direct interaction and/or indirect contributions. Indirect contributions included the construction of the overall protein structure, especially the cap-binding pocket.Keywords: human eIF-4E ; m'G(5')ppp ; mRNA cap structure ; site-directed mutagenesis ; cap binding.Many eukaryotic mRNAs have a common cap structure [m7G(5')ppp(5')N, where N is any nucleotide] at the 5'-terminal portion and its structure plays important roles in stabilizing the mRNA structure and facilitating mRNA binding to ribosomes during initiation [l-31. To allow for the efficient translation of mRNA, however, an interaction is required between the cap structure and eukaryotic initiation factor (eIF)-4 polypeptides consisting of eIF-4A, eIF-4B, eIF-4E, and eIF-4y 143. eIF-4E, which corresponds to the smallest subunit of eIF-4F, has been shown to bind specifically to the mRNA cap structure and has an important function in the first step of protein synthesis; it appears to play a key role in the regulation of translation via phosphorylation [5 -71. Furthermore, recent studies have indicated that eIF-4E participates in the regulation of translation through the interaction with 4E-BP [8].The amino acid sequences of eIF-4Es from yeast [9], human [lo], mouse [ I l l , rabbit 1121, wheat germ [13], and Drosophila [14] have already been reported. Human eIF-4E is a polypeptide of about 25 kDa that contains eight tryptophan residues and its amino acid residues have been remarkably conserved in number and position during evolution among human, mouse, yeast, rabbit, wheat, and Drosophila, which probably reflects the importance for the recognition of the mRNA cap structure [15]. From fluorescence studies at various pH values, however, histidineCorrespondt.nce to T. Ishida, Department of Physical Chemistry, Osaka University of Pharmaceutical Scie...

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