Characterization of the two eIF4A-binding sites on human eIF4G-1

Area of Science:

• Molecular Biology
• Protein-RNA Interactions

Background:

• Eukaryotic translation initiation factor 4G-1 (eIF4G) is essential for mRNA recruitment to the 43 S preinitiation complex.
• eIF4G possesses two distinct binding sites for the RNA helicase eIF4A: one in the central domain and another in the COOH-terminal domain.

Purpose of the Study:

• To investigate the binding stoichiometry and kinetics of eIF4A to eIF4G.
• To determine if eIF4A can bind to both sites on eIF4G simultaneously.
• To characterize the cooperative binding of eIF4A to eIF4G.

Main Methods:

• Utilized recombinant eIF4G fragments to study eIF4A binding.
• Employed surface plasmon resonance (SPR) to determine binding kinetics (association and dissociation rate constants) and affinity (dissociation equilibrium constant).
• Investigated the effect of eIF3 on eIF4A binding to eIF4G.

Main Results:

• Fragments with single eIF4A binding sites exhibited 1:1 stoichiometry, while fragments with both sites showed 1:2 stoichiometry.
• Cooperative binding of eIF4A to eIF4G was observed, with higher binding to the dual-site fragment than predicted by summing single-site bindings.
• Surface plasmon resonance revealed distinct binding kinetics for the central (Ka: 1.2 x 10^5 M^-1 s^-1, Kd: 2.1 x 10^-3 s^-1, KD: 17 nM) and COOH-terminal (Ka: 5.1 x 10^3 M^-1 s^-1, Kd: 1.7 x 10^-3 s^-1, KD: 330 nM) sites.
• Competition assays indicated that a single eIF4A molecule cannot bind to both sites on eIF4G concurrently.

Conclusions:

• eIF4G binds eIF4A with a 1:2 stoichiometry, demonstrating cooperative binding.
• The two eIF4A binding sites on eIF4G are functionally distinct with different affinities and kinetics.
• Despite cooperative binding, simultaneous binding of a single eIF4A molecule to both sites is not possible.