Interaction of ferritin iron responsive element (IRE) mRNA with translation initiation factor eIF4F

The interaction of ferritin iron responsive element (IRE) mRNA with eIF4F was examined by fluorescence and circular dichroism spectroscopy. Fluorescence quenching data indicated that eIF4F contains one high affinity binding site for ferritin IRE RNA. The Scatchard analysis revealed strong binding affinity (K_a = 11.1 × 10⁷ M^-1) and binding capacity (n = 1.0) between IRE RNA and eIF4F. The binding affinity of IRE RNA for eIF4F decreased (~4-fold) as temperature increased (from 5 °C to 30 °C). The van't Hoff analysis revealed that IRE RNA binding to eIF4F is enthalpy-driven (ΔH = -47.1 ± 3.4 kJ/mol) and entropy-opposed (ΔS = -30.1 ± 1.5 J/mol/K). The addition of iron increased the enthalpic, while decreasing the entropic contribution towards the eIF4F•IRE RNA complex, resulting in favorable free energy (ΔG = -49.8 ± 2.8 kJ/mol). Thermodynamic values and ionic strength data suggest that the presence of iron increases hydrogen bonding and decreases hydrophobic interactions, leading to formation of a more stable complex. The interaction of IRE RNA with eIF4F at higher concentrations produced significant changes in the secondary structure of the protein, as revealed from the far-UV CD results, clearly illustrating the structural alterations resulted from formation of the eIF4F•IRE RNA complex. A Lineweaver-Burk plot showed an uncompetitive binding behavior between IRE RNA and m⁷G cap for the eIF4F, indicating that there are different binding sites on the eIF4F for the IRE RNA and the cap analog; molecular docking analysis further supports this notion. Our findings suggest that the eIF4F•IRE RNA complex formation is accompanied by an elevated hydrogen bonding and weakened hydrophobic interactions, leading to an overall conformational change, favored in terms of its free energy. The conformational change in the eIF4F structure, caused by the IRE RNA binding, provides a more stable platform for effective IRE translation in iron homeostasis.