Abstract
Eukaryotic translation initiation involves two conserved DEAD-box RNA helicases, eIF4A and Ded1p. Here we show that S. cerevisiae eIF4A and Ded1p directly interact with each other and simultaneously with the scaffolding protein eIF4G. We delineate a comprehensive thermodynamic framework for the interactions between Ded1p, eIF4A, eIF4G, RNA and ATP, which indicates that eIF4A, with and without eIF4G, acts as a modulator for activity and substrate preferences of Ded1p, which is the RNA remodeling unit in all complexes. Our results reveal and characterize an unexpected interdependence between the two RNA helicases and eIF4G, and suggest that Ded1p is an integral part of eIF4F, the complex comprising eIF4G, eIF4A, and eIF4E.
Highlights
Translation initiation in eukaryotes involves at least 12 distinct protein factors, including two highly conserved DEAD-box rate constants (RNA) helicases, eIF4A and Ded1p (DDX3 in human) (Aitken and Lorsch, 2012; Hinnebusch, 2014; Parsyan et al, 2011)
Ded1-95 bears a T408I mutation, which impairs RNA binding by Ded1p in vitro (Figure 1—figure supplement 1)
Our model further revealed that unwinding rate constants at ATP and protein saturation and RNA affinities at ATP saturation are similar for the Ded1p trimer and for the complexes containing Ded1p and eIF4A (Figure 5C, Supplementary file 2A)
Summary
Translation initiation in eukaryotes involves at least 12 distinct protein factors, including two highly conserved DEAD-box RNA helicases, eIF4A and Ded1p (DDX3 in human) (Aitken and Lorsch, 2012; Hinnebusch, 2014; Parsyan et al, 2011). EIF4A unwinds RNA duplexes in vitro (Merrick, 2015). The helicase interacts with the large scaffolding protein eIF4G that binds the cap-binding protein eIF4E (Hinnebusch,2014; Parsyan et al, 2011). EIF4A is thought to be critical for steps during and subsequent to the recruitment of the 40S ribosome subunit to the mRNA (Parsyan et al, 2011). In S. cerevisiae, eIF4A appears to promote at least one step necessary for the translation of most or all mRNAs (Sen et al, 2015)
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