Journal: Molecular and Cellular Biology
Article Title: Eukaryotic Translation Initiation Factor 4E (eIF4E) Binding Site and the Middle One-Third of eIF4GI Constitute the Core Domain for Cap-Dependent Translation, and the C-Terminal One-Third Functions as a Modulatory Region
Figure Lengend Snippet: Functional analysis of eIF4GI deletion mutants. (A) Schematic representation of eIF4GI deletion mutants. PABP, eIF4E, eIF4A, eIF3, and Mnk1 binding sites are indicated. (B) Toeprint analysis of 48S ribosomal complex formation on β-globin mRNA with eIF4GI deletion mutants. The reaction components are indicated above the lanes. The value for eIF4GI(157–1560) (lane 3) was set at 100. (C) Analysis of eIF4GI deletion mutants in a reticulocyte lysate translation system. Translation was performed as described in Materials and Methods. A rabbit reticulocyte lysate treated with rhinovirus 2A pro was supplemented with recombinant proteins as indicated and programmed for translation with the capped bicistronic mRNA CAT/EMCV IRES/LUC. For quantitation of luciferase (LUC) synthesis, the value obtained for translation in untreated lysate in the absence of additional proteins (lane 1) was set at 100. For the quantitation of CAT synthesis, the value obtained for translation in the treated lysate in the presence of eIF4E alone was subtracted as background, and then the value for treated lysate translated in the presence of eIF4E and eIF4GI(157–1560) (lane 4) was set at 100. (D) Western blotting of eIF4G deletion mutants. Recombinant protein preparations (∼1 μg) containing the same amount of eIF4GI according to Coomassie blue staining were subjected to SDS-PAGE (10% gel) and analyzed by Western blotting with anti-Xpress antibody (to detect the epitope located between the His tag and eIF4G coding sequence) or with anti-FLAG antibody.
Article Snippet: Reaction mixtures (40 μl) containing native α- and β-globin mRNA (Life Technologies) (0.3 μg), His-eIF1 (0.5 μg), His-eIF1A (0.5 μg), eIF2 (3 μg), eIF3 (7 μg), FLAG-eIF4B (1 μg), Met-tRNAi Met (4 pmol), 40S ribosomal subunits (4 pmol), His- or FLAG-eIF4GI (2 μg, unless indicated), His-eIF4E (0.3 μg), and His-eIF4A (3 μg) were incubated in buffer (2 mM dithiothreitol, 100 mM KCl, 20 mM Tris-HCl [pH 7.6], 2.5 mM magnesium acetate, 100 U of RNasin [Promega], 1 mM ATP, 0.4 mM guanylyl imidodiphosphate [GMP-PNP], 250 μM spermidine) for 5 min at 30°C.
Techniques: Functional Assay, Binding Assay, Recombinant, Quantitation Assay, Luciferase, Western Blot, Staining, SDS Page, Sequencing