Binding analysis of Xenopus laevis translation initiation factor 4E (eIF4E) in initiation complex formation

A translation initiation factor, eIF4E, of Xenopus laevis was purified by a ffinity column chromatography after the gene expression as a full-length pr otein in a baculovirus-insect cell system. Interaction between X, laevis eI F4E and 4E-BP2 was analyzed by affinity column chromatography, gel permeati on chromatography (GPC), and surface plasmon resonance (SPR), It was found that the interaction of eIF4E with an mRNA cap-analogue enhanced the bindin g activity of eIF4E with 4E-BP2, Furthermore, the SPR analysis showed that the eIF4E-cap-analogue interaction was very weak regardless of complex form ation of 4E-BP2 with eIF4E; the dissociation constant of eIF4E for the cap- analogue was estimated to be 10(-2)-10(-4) M, These results suggest that th e participation of another initiation factor is required for eIF4E to recog nize the cap structure in vivo. The results reported in this paper support "the performed complex model" of Lee et al,, in which eIF4E binds to the mR NA cap structure after the initiation factors have formed the initiation co mplex eIF4F.