Human eukaryotic initiation factor 4E (eIF4E) and the nucleotide-bound state of eIF4A regulate eIF4F binding to RNA.

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• The mechanism of eukaryotic translation initiation factor 4F (eIF4F) complex assembly and mRNA binding is not fully understood.
• eIF4F, composed of eIF4E, eIF4A, and eIF4G, plays a crucial role in cap-dependent translation initiation.
• Understanding the dynamics of eIF4F-mRNA interaction is key to deciphering translation regulation.

Purpose of the Study:

• To elucidate the coordination between eIF4E, eIF4A, and eIF4G in regulating eIF4F binding to mRNA.
• To establish thermodynamic and kinetic frameworks for uncapped RNA interaction with human eIF4F.
• To investigate the role of ATP in modulating eIF4F-RNA dynamics.

Main Methods:

• Fluorescence anisotropy was employed to study the binding kinetics and thermodynamics.
• Real-time monitoring of uncapped RNA dissociation from eIF4F was performed.
• Kinetic analysis of eIF4A binding to eIF4G was conducted.

Main Results:

• eIF4E binding to eIF4G induces a high-affinity RNA-binding conformation of eIF4F.
• ATP binding to eIF4A significantly enhances eIF4F's affinity for uncapped RNA by reducing dissociation rates.
• eIF4F exhibits rapid association and dissociation rates with RNA upon ATP release from eIF4A.
• Two distinct kinetic states of eIF4A binding to eIF4G suggest conformational flexibility within the eIF4F complex.
• The eIF4G autoinhibitory domain stabilizes eIF4A binding, but this is overcome by eIF4E.

Conclusions:

• eIF4E binding to eIF4G stabilizes a high-affinity RNA-binding state of eIF4F.
• eIF4A's dynamic interaction with eIF4G, regulated by ATP, facilitates rapid mRNA binding and release during scanning.
• The findings provide a model for eIF4F's dynamic role in translation initiation.