Journal: Nucleic Acids Research

Article Title: Synergistic activation of eIF4A by eIF4B and eIF4G

doi: 10.1093/nar/gkq1206

Figure Lengend Snippet: Interactions between eIF4A, eIF4B and eIF4G fragments. ( A ) Schematic representation of the constructs used in this study or mentioned in the text. Colored bars show motifs expected to be involved in protein–protein interactions (interacting partner indicated) and motifs involved in dimerization (DRYG) or RNA binding (RRM and ARM). Poly(A) binding protein (PAPB). yeIF4G is the yeast homolog of mammalian eIF4G. The numbering in parenthesis for yeIF4G and eIF4GII correspond to the amino acid position, while the numbering above refer to the corresponding position in human eIF4GI based on sequence alignment using Clustal W. ( B ) Cobalt pull-down experiments using eIF4A-His 6 (lanes 1–6) or negative controls without (lanes 7–10) and the indicated proteins. Proteins eluted using 400 mM Imidazole from the TALON beads are shown in the upper gel (eluted) and 15% of the flow through (FT) is shown in the lower gel. All eIF4A-His 6 were bound to the TALON beads. ( C ) Streptavidin pull-down experiments using a pU 30 biotinylated RNA. All reactions contain eIF4B and eIF4A and where indicated His 6 -eIF4G-MC, nucleotide analogs or biotinylated RNA. All reactions were incubated for 1 h at 4°C. The upper gel shows the proteins that bound to the RNA (boiled) and the lower gel shows 15% of the flow through (FT). ( D ) As in (C) but reactions contain eIF4A, eIF4BΔC and pU 30 -biotin along with the indicated amount of eIF4G-MC and ADP-AlF x as nucleotide analog. Each reaction was incubated at 4°C for the indicated time (min). ( E ) As in (D) but using fixed amount of either eIF4G-MC, eIF4G-M or eIF4G-C where indicated. Each reaction was incubated for 1 hr at room temperature. The asterisk indicates the position of the streptavidin moiety eluted by boiling. Due to the similar position of eIF4G-MC and eIF4B on a SDS–PAGE gel, eIF4BΔC was used in (B), (D) and (E). Note that eIF4B has a strong nucleotide-independent RNA binding leading to a basal level of eIF4B while eIF4G-MC has a low nucleotide-independent RNA binding. eIF4A has a low nucleotide-dependent RNA binding.

Article Snippet: Cobalt charged TALON beads (TALON Superflow, BD Bioscience) were prepared (20 μl of a 50% slurry per reaction) by washing three times with 50 μl buffer A (150 mM NaCl, 20 mM Tris–HCl pH 7.6, 5 mM MgCl 2 , 10% glycerol, 0.1% NP40, 0.5 mM β-ME) per reaction.

Techniques: Construct, RNA Binding Assay, Binding Assay, Sequencing, Incubation, SDS Page