Abstract
Barley yellow dwarf virus RNA, lacking a 5' cap and a 3' poly(A) tail, contains a cap-independent translation element (BTE) in the 3'-untranslated region that interacts with host translation initiation factor eIF4G. To determine how eIF4G recruits the mRNA, three eIF4G deletion mutants were constructed: (i) eIF4G601-1196, containing amino acids 601-1196, including the putative BTE-binding region, and binding domains for eIF4E, eIF4A, and eIF4B; (ii) eIF4G601-1488, which contains an additional C-terminal eIF4A-binding domain; and (iii) eIF4G742-1196, which lacks the eIF4E-binding site. eIF4G601-1196 binds BTE tightly and supports efficient translation. The helicase complex, consisting of eIF4A, eIF4B, and ATP, stimulated BTE binding with eIF4G601-1196 but not eIF4G601-1488, suggesting that the eIF4A binding domains may serve a regulatory role, with the C-terminal binding site having negative effects. eIF4E binding to eIF4G601-1196 induced a conformational change, significantly increasing the binding affinity to BTE. A comparison of the binding of eIF4G deletion mutants with BTEs containing mutations showed a general correlation between binding affinity and ability to facilitate translation. In summary, these results reveal a new role for the helicase complex in 3' cap-independent translation element-mediated translation and show that the functional core domain of eIF4G plus an adjacent probable RNA-binding domain mediate translation initiation.
Highlights
Barley yellow dwarf virus RNA, lacking a 5 cap and a 3 poly(A) tail, contains a cap-independent translation element (BTE) in the 3-untranslated region that interacts with host translation initiation factor eukaryotic translation initiation factor 4G (eIF4G)
The helicase complex, consisting of eIF4A, eIF4B, and ATP, stimulated BTE binding with eIF4G601–1196 but not eIF4G601–1488, suggesting that the eIF4A binding domains may serve a regulatory role, with the C-terminal binding site having negative effects. eIF4E binding to eIF4G601– 1196 induced a conformational change, significantly increasing the binding affinity to BTE
EIF4F exists as a heterodimer composed of eIF4E and eukaryotic translation initiation factor 4G [4]. eIF4G serves as a scaffolding protein to recruit other translation initiation factors such as eIF4A, eIF4B, and poly(A)-binding protein (PABP) [5, 6]
Summary
In eIF4F-depleted wheat germ extract [21, 26]. In this study, the translation stimulation efficiencies of three eIF4G deletion mutants were determined. eIF4G601–1196 is the eIF4G fragment containing amino acid residues 601–1196, including the eIF4E binding site (aa 710 –721), one eIF4A binding site, one eIF4B binding site, and a region required for BTE binding (aa 766 – 863). eIF4G601–1488 is a longer fragment with one additional C-terminal eIF4A binding domain (aa 1300 –1488); eIF4G742–1196 is a shorter deletion mutant lacking the eIF4E binding sequence, as well as the second eIF4A site (Fig. 1B). The eIF4G fragment lacking the eIF4E binding site (eIF4G742–1196) restored BlucB translation to 63%Ϯ10% of level obtained with eIF4F (Fig. 2B), confirming that the eIF4E-interacting region of eIF4G is unnecessary. EIF4G742– 1196, which lacks the eIF4E binding region, had a weaker binding affinity to the BTE (KD 91 Ϯ 12 nM) compared with other two eIF4G deletion mutants (Table 1 and Fig. 3). When eIF4A and eIF4B were added along with eIF4F or eIF4G mutants into the eIF4F-depleted WGE, BlucB translation was at least double the level obtained in the absence of eIF4A and eIF4B after 40 min (Fig. 2D). The equilibrium dissociation constant between eIF4G601–1196 and BTE decreased (29 Ϯ 1 nM compared with 40 Ϯ 4 nM) (Table 1), indicating that in addition to possible RNA unwinding, binding is affected by protein-protein interactions between eIF4A, eIF4B, and eIF4G. 40 Ϯ 4 59 Ϯ 6 79 Ϯ 2 127 Ϯ 11 13 Ϯ 2 a The binding affinity has not been determined
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