Analysis of the mRNA cap-binding ability of human eukaryotic initiation factor-4E by use of recombinant wild-type and mutant forms.
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 mutagenesis, 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.
Morino
S
S
Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, Japan.
Hazama
H
H
Ozaki
M
M
Teraoka
Y
Y
Shibata
S
S
Doi
M
M
Ueda
H
H
Ishida
T
T
Uesugi
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D039561:Eukaryotic Initiation Factor-4E; D005813:Genes, Synthetic; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012314:RNA Cap Analogs / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship
D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012314:RNA Cap Analogs; D012315:RNA Caps; D011994:Recombinant Proteins; C017516:7-methylguanosine triphosphate