Characterization of the two eIF4A-binding sites on human eIF4G-1.
Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. eIF4G has two binding sites for the RNA helicase eIF4A, one in the central domain and one in the COOH-terminal domain. Recombinant eIF4G fragments that contained each of these sites separately bound eIF4A with a 1:1 stoichiometry, but fragments containing both sites bound eIF4A with a 1:2 stoichiometry. eIF3 did not interfere with eIF4A binding to the central site. Interestingly, at the same concentration of free eIF4A, more eIF4A was bound to an eIF4G fragment containing both eIF4A sites than the sum of binding to fragments containing the single sites, indicating cooperative binding. Binding of eIF4A to an immobilized fragment of eIF4G containing the COOH-terminal site was competed by a soluble eIF4G fragment containing the central site, indicating that a single eIF4A molecule cannot bind simultaneously to both sites. The association rate constant, dissociation rate constant, and dissociation equilibrium constant for each site were determined by surface plasmon resonance and found to be, respectively, 1.2 x 10(5) m(-1) s(-1), 2.1 x 10(-3) s(-1), and 17 nm for the central site and 5.1 x 10(3) m(-1) s(-1), 1.7 x 10(-3) s(-1), and 330 nm for the COOH-terminal site.
Korneeva
N L
NL
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport 71130-3932, USA.
Lamphear
B J
BJ
Hennigan
F L
FL
Merrick
W C
WC
Rhoads
R E
RE
D001665:Binding Sites; D001667:Binding, Competitive; D039601:Eukaryotic Initiation Factor-4A; D039603:Eukaryotic Initiation Factor-4G; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3; D011485:Protein Binding; D011994:Recombinant Proteins; D020349:Surface Plasmon Resonance
C113920:EIF4G1 protein, human; D039603:Eukaryotic Initiation Factor-4G; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D039665:Prokaryotic Initiation Factor-3; D011994:Recombinant Proteins; D039601:Eukaryotic Initiation Factor-4A