Therefore, we used this e IF4GI truncation in subsequent experiments.We next examined whether AUG initiation codon selection affects e IF3j-Fl affinity during m RNA recruitment.Taken together, these studies suggest a model in which a full accommodation of m RNA in the m RNA entry channel of the 40S subunit corresponds to a reduced affinity of e IF3j for the 40S subunit.This model has allowed us to exploit the change in e IF3j affinity for the 43S PIC to quantitatively monitor the process of m RNA recruitment.
However, beyond the established function of its cap-binding (e IF4E), helicase (e IF4A), and e IF3-binding (e IF4G) components, very little is known regarding the molecular mechanism by which e IF4F promotes these stages of initiation.
In this study, we demonstrate that the reduction in e IF3j affinity for the 40S subunit is unexpectedly dependent on both the e IF4A DEAD-box helicase and ATP.
Importantly, we show that this function of e IF4A is completely independent of its unwinding activity and likely constitutes a new regulated step in m RNA recruitment..
Unexpectedly, we show that a full reduction in e IF3j affinity for the 43S PIC requires an ATP-dependent, but unwinding-independent, activity of e IF4A.
This result suggests that in addition to its helicase activity, e IF4A uses the free energy of ATP binding and hydrolysis as a regulatory switch to control the conformation of the 43S PIC during m RNA recruitment.