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 affinity column chromatography after the gene expression as a full-length protein in a baculovirus-insect cell system. Interaction between X. laevis eIF4E and 4E-BP2 was analyzed by affinity column chromatography, gel permeation chromatography (GPC), and surface plasmon resonance (SPR). It was found that the interaction of eIF4E with an mRNA cap-analogue enhanced the binding activity of eIF4E with 4E-BP2. Furthermore, the SPR analysis showed that the eIF4E-cap-analogue interaction was very weak regardless of complex formation 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 the participation of another initiation factor is required for eIF4E to recognize 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 mRNA cap structure after the initiation factors have formed the initiation complex eIF4F.
Miyoshi
H
H
Institute for Biomolecular Science, Gakushuin University, Toshima-ku, Tokyo 171-8588, Japan.
Youtani
T
T
Ide
H
H
Hori
H
H
Okamoto
K
K
Ishikawa
M
M
Wakiyama
M
M
Nishino
T
T
Ishida
T
T
Miura
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D002352:Carrier Proteins; C104151:EIF4EBP2 protein, human; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; D012315:RNA Caps; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins