Synergistic activation of eIF4A by eIF4B and eIF4G

• Published in: Nucleic acids research 2011-04, Vol.39 (7), p.2678-2689
• Main Authors: Nielsen, Klaus H, Behrens, Manja A, He, Yangzi, Oliveira, Cristiano L P, Jensen, Lars Sottrup, Hoffmann, Søren V, Pedersen, Jan S, Andersen, Gregers R
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Eukaryotic Initiation Factor-4A - chemistry; Eukaryotic Initiation Factor-4A - metabolism; Eukaryotic Initiation Factor-4G - chemistry; Eukaryotic Initiation Factor-4G - metabolism; Eukaryotic Initiation Factors - metabolism; Molecular Biology; Protein Binding; Scattering, Small Angle; X-Ray Diffraction
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkq1206

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.