Abstract
Translation initiation factor eIF4F (eukaryotic initiation factor 4F), composed of eIF4E, eIF4G, and eIF4A, binds to the m(7)G cap structure of mRNA and stimulates recruitment of the 43S preinitiation complex and subsequent scanning to the initiation codon. The HEAT domain of eIF4G stabilizes the active conformation of eIF4A required for its RNA helicase activity. Mammalian eIF4B also stimulates eIF4A activity, but this function appears to be lacking in yeast, making it unclear how yeast eIF4B (yeIF4B/Tif3) stimulates translation. We identified Ts(-) mutations in the HEAT domains of yeast eIF4G1 and eIF4G2 that are suppressed by overexpressing either yeIF4B or eIF4A, whereas others are suppressed only by eIF4A overexpression. Importantly, suppression of HEAT domain substitutions by yeIF4B overexpression was correlated with the restoration of native eIF4A·eIF4G complexes in vivo, and the rescue of specific mutant eIF4A·eIF4G complexes by yeIF4B was reconstituted in vitro. Association of eIF4A with WT eIF4G in vivo also was enhanced by yeIF4B overexpression and was impaired in cells lacking yeIF4B. Furthermore, we detected native complexes containing eIF4G and yeIF4B but lacking eIF4A. These and other findings lead us to propose that yeIF4B acts in vivo to promote eIF4F assembly by enhancing a conformation of the HEAT domain of yeast eIF4G conducive for stable binding to eIF4A.
Highlights
Mammalian eIF4B stimulates eIF4A helicase activity, but its function in promoting translation initiation in yeast is unclear
Because the TIF4632 allele of interest harbored multiple mutations, site-directed mutagenesis was employed to generate a panel of alleles with single mutations. The outcome of this analysis was that the tif4632-L574F mutation was found to be necessary and sufficient for the TsϪ phenotype displayed by the original compound mutation, and, importantly, the TsϪ phenotype conferred by L574F is mitigated by overexpressing either yeIF4B or eIF4A in the mutant cells (Fig. 1A, hc TIF3 and hc TIF1 versus vector, 36 °C)
We demonstrated that various point mutations in the HEAT domains of eIF4G1 and eIF4G2 that weaken the binding to eIF4A and are suppressed in vivo by overexpressing eIF4A are suppressed by overexpressing yeIF4B
Summary
Mammalian eIF4B stimulates eIF4A helicase activity, but its function in promoting translation initiation in yeast is unclear. We detected native complexes containing eIF4G and yeIF4B but lacking eIF4A These and other findings lead us to propose that yeIF4B acts in vivo to promote eIF4F assembly by enhancing a conformation of the HEAT domain of yeast eIF4G conducive for stable binding to eIF4A. Initiation factors (the 43S PIC) to the 5Ј-untranslated region (5Ј-UTR) of the mRNA This reaction is stimulated by eIF4F, bound to the m7G cap structure of the mRNA, composed of cap-binding protein eIF4E, eIF4G, and the DEAD-box RNA helicase eIF4A. Beyond stimulating eIF4A helicase activity, it was suggested that mammalian eIF4B can promote ribosome attachment more directly by binding simultaneously to mRNA and 18S rRNA through its multiple RNA-binding domains [21] or by forming a protein bridge between the eIF4F1⁄7mRNP and eIF3 in the PIC [22]. Our results suggest that one aspect of yeIF4B function is to promote binding of eIF4A to the eIF4G1⁄7eIF4E subassembly of eIF4F with attendant recruitment of eIF4A to the cap structure of mRNA to promote 43S PIC attachment and subsequent scanning for the start codon
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