Journal: Journal of Virology
Article Title: Interaction of Translation Initiation Factor eIF4B with the Poliovirus Internal Ribosome Entry Site
Figure Lengend Snippet: Detection of eIF4B in ribosomal initiation complexes with poliovirus IRES RNA. (A) Radioactivity profile of the gradients. [α- 32 P]CTP-labeled poliovirus IRES 1-745 RNA was incubated with RRL for 10 min at 30°C either in the absence of translation inhibitors (solid line), in the presence of 4 mM GMP-PNP (broken line), or in the presence of 0.17 mM anisomycin (dotted line). Samples were irradiated with UV, and initiation complexes were separated on a 10 to 35% sucrose gradient. Fractions were collected from the bottom (fraction 1), and aliquots were used for scintillation counting. The amount of radioactivity in each fraction relative to the total amount of radioactivity in the entire gradient (% cpm) is plotted versus the fraction number. The ribosomal 80S and 48S peaks are indicated. (B and C) Identification of ribosomal subunits in gradients run in parallel with the gradients in panel A. (B) Nucleic acids isolated from gradient fractions were heated, separated on an 1% agarose gel, and stained with ethidium bromide. The positions of 18S and 28S rRNAs from a preparation of ribosomal RNAs (R) are labeled on the left. (C) Immunoblot of gradient fractions with an antibody directed against ribosomal protein S6. (D) UV cross-linked proteins from the gradients shown in panel A. Fractions were treated with RNase A, and proteins were precipitated with TCA, resolved on SDS-polyacrylamide gels, and visualized by autoradiography. Molecular masses of marker proteins (M) are given in kilodaltons. The position of eIF4B is indicated by arrows on the right.
Article Snippet: Labeled RNAs were synthesized as described previously ( ) by using SP6 RNA polymerase in the presence of 2.5 μM [α-32 P]CTP, -ATP, -GTP, or -UTP (400 Ci/mmol; Amersham) as indicated with 15 μM nonradioactive labeling nucleotide added.
Techniques: Radioactivity, Labeling, Incubation, Irradiation, Isolation, Agarose Gel Electrophoresis, Staining, Autoradiography, Marker