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.
Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus, Denmark. khn@mb.au.dk
Klaus H
KH
Nielsen
Manja A
MA
Behrens
Yangzi
Y
He
Cristiano L P
CL
Oliveira
Lars Sottrup
LS
Jensen
Søren V
SV
Hoffmann
Jan S
JS
Pedersen
Gregers R
GR
Andersen
D000251:Adenosine Triphosphatases / Q000378:metabolism; D039601:Eukaryotic Initiation Factor-4A / Q000737:chemistry / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G / Q000737:chemistry / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000378:metabolism*; D011485:Protein Binding; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
D039603:Eukaryotic Initiation Factor-4G; D039642:Eukaryotic Initiation Factors; C031799:eIF-4B; D039601:Eukaryotic Initiation Factor-4A; D000251:Adenosine Triphosphatases