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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.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.To this end, a reconstituted human 43S PIC was incubated with saturating amounts of m RNA, the cap-binding complex e IF4F together with ATP (e IF4F-ATP), and the accessory protein e IF4B.A high-affinity binding of fluorescent-labeled e IF3j (e IF3j-Fl) was confirmed within the 43S PIC, as reported previously (12) (; anisotropy data are summarized in Table S1).This function of e IF4A is completely independent of its unwinding activity and thus functions as a regulator of translation initiation regardless of m RNA secondary structure content.
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.
Importantly, that study showed that a short m RNA that does not extend into the entry channel fails to displace e IF3j.
A similar observation was also found for initiation mediated by the hepatitis C virus internal ribosome entry site, where an m RNA truncated after the initiation codon failed to displace e IF3j (11).
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..
Before the recruitment of the m RNA, the 40S subunit is bound by eukaryotic initiation factor (e IF) 1, e IF1A, e IF2-GTP-Met-t RNA, e IF3, and e IF5 to form the 43S preinitiation complex (PIC) (1).