The domains of yeast eIF4G, eIF4E and the cap fine-tune eIF4A activities through an intricate network of stimulatory and inhibitory effects

Translation initiation in eukaryotes starts with the recognition of the mRNA 5′-cap by eIF4F, a hetero-trimeric complex of eIF4E, the cap-binding protein, eIF4A, a DEAD-box helicase, and eIF4G, a scaffold protein. eIF4G comprises eIF4E- and eIF4A-binding domains (4E-BD, 4A-BD) and three RNA-binding regions (RNA1–RNA3), and interacts with eIF4A, eIF4E, and with the mRNA. Within the eIF4F complex, the helicase activity of eIF4A is increased. We showed previously that RNA3 of eIF4G is important for the stimulation of the eIF4A conformational cycle and its ATPase and helicase activities. Here, we dissect the interplay between the eIF4G domains and the role of the eIF4E/cap interaction in eIF4A activation. We show that RNA2 leads to an increase in the fraction of eIF4A in the closed state, an increased RNA affinity, and faster RNA unwinding. This stimulatory effect is partially reduced when the 4E-BD is present. eIF4E binding to the 4E-BD then further inhibits the helicase activity and closing of eIF4A, but does not affect the RNA-stimulated ATPase activity of eIF4A. The 5′-cap renders the functional interaction of mRNA with eIF4A less efficient. Overall, the activity of eIF4A at the 5′-cap is thus fine-tuned by a delicately balanced network of stimulatory and inhibitory interactions.