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

During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. In addition, we show that the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation con-stant observed in the presence versus the absence of ATP. Monitoring uncapped RNA dissociation in real time reveals that ATP reduces the dissociation rate constant of RNA for eIF4F by ,-,4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a dynamic state that has very fast asso-ciation and dissociation rates from RNA. Monitoring the ki-netic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined that the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A-binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.

Automated summary

This study reveals the mechanism by which eukaryotic initiation factors regulate the binding of eIF4F to mRNA during translation initiation. The researchers found that eIF4E binding to eIF4G generates a high-affinity conformation of the eIF4F complex for RNA, and the nucleotide-bound state of eIF4A further regulates RNA binding. They also observed that the autoinhibitory domain of eIF4G promotes a stable eIF4A-binding state, which is overcome by eIF4E binding. These findings provide insights into the dynamic interaction between eIF4F and mRNA during the scanning process.