Synergistic activation of eIF4A by eIF4B and eIF4G

eIF4A is a key component in eukaryotic translation initiation; however, it has not been clear how auxiliary factors like eIF4B and eIF4G stimulate eIF4A and how this contributes to the initiation process. Based on results from isothermal titration calorimetry, we propose a two-site model for eIF4A binding to an 83.5 kDa eIF4G fragment (eIF4G-MC), with a high- and a low-affinity site, having binding constants KD of ∼50 and ∼1000 nM, respectively. Small angle X-ray scattering analysis shows that the eIF4G-MC fragment adopts an elongated, well-defined structure with a maximum dimension of 220 Å, able to span the width of the 40S ribosomal subunit. We establish a stable eIF4A–eIF4B complex requiring RNA, nucleotide and the eIF4G-MC fragment, using an in vitro RNA pull-down assay. The eIF4G-MC fragment does not stably associate with the eIF4A–eIF4B–RNA-nucleotide complex but acts catalytically in its formation. Furthermore, we demonstrate that eIF4B and eIF4G-MC act synergistically in stimulating the ATPase activity of eIF4A.

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