[Gene expression of human eukaryotic initiation factor-4E for protein synthesis and study of its recognition mechanism of mRNA cap structure]

Abstract

Being stimulated by the insights from model studies that (i) the intimate combination of hydrogen-bonding pairing and aromatic stacking interactions is important for the specific binding of guanine base by peptide and (ii) the pi-pi stacking force of Trp is significantly strengthened by the guanine N7-methylation (m7G), this research project was started, because (a) the mRNA cap structure is characterized by the existence of m7G and (b) an eukaryotic initiation factor-4E (eIF-4E), a protein which specifically recognizes the mRNA cap structure and opens the protein biosynthesis, contains 8 Trp residues irrespective of its relatively low molecular weight of about 25 kDa. In order to prepare the sufficient amount of sample for carrying out the analysis of the recognition mechanism of mRNA cap structure by eIF-4E at the atomic level, firstly, the expression of human eIF-4E gene in Escherichia coli was attempted. An artificial gene encoding for human eIF-4E was chemically synthesized and succeeded in the expression with two different forms, i.e., as a fusion protein with human growth hormone and a direct expression of soluble protein. The isolation of eIF-4E and its purification procedure using the m7GTP affinity chromatography were accomplished. It was shown by spectroscopic methods that the recombinant eIF-4E exhibits essentially the same tertiary structure as the native one and the binding ability with mRNA cap analog was identical with each other. In order to analyze the functional amino acid residues which are essential for specific recognition of mRNA cap structure, next a series of eIF-4E mutants were prepared by the site-directed mutagenesis, and His37, His200, Trp102 and Glu103 were suggested to be important for binding of mRNA cap structure, as judged from comparison of the binding abilities of respective mutants with a m7GTP affinity column. Since the crystals of recombinant eIF-4E-m7GTP complex suitable for X-ray crystallography are now in preparation, the detailed interaction mode between them will be opened in near future.