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 tw o binding sites for the RNA helicase eIF4A, one in the central domain and o ne in the COOH-terminal domain. Recombinant eIF4G fragments that contained each of these sites separately bound eIF4A with a 1:1 stoichiometry, but fr agments containing both sites bound eIF4A with a 1:2 stoichiometry. eIF3 di d not interfere with eIF4A binding to the central site. Interestingly, at t he same concentration of free eIF4A, more eIF4A was bound to an eIF4G fragm ent containing both eIF4A sites than the sum of binding to fragments contai ning the single sites, indicating cooperative binding. Binding of eIF4A to an immobilized fragment of eIF4G containing the COOH-terminal site was comp eted by a soluble eIF4G fragment containing the central site, indicating th at a single eIF4A molecule cannot bind simultaneously to both sites. The as sociation rate constant, dissociation rate constant, and dissociation equil ibrium 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.