Figure 8.

Activities of eIF1, IF3, IF3-CTD and YciH in discriminating against initiation on the CrPV IGR IRES. (A, B) Toe-printing analysis of binding of 40S, 30S subunits, 80S and 70S ribosomes to this IRES in the presence of eIF1, IF3, IF3-CTD, YciH, and eEF1, eEF2 and Ala-tRNA^Ala as indicated. The 40S/80S-dependent toe-prints and toe-prints that appear following ribosomal translocation are indicated to the right. Full-length cDNAs are labeled. (C) Influence of IF3 on translation mediated by the CrPV IRES. In total, 0.2 μg of bicistronic RLuc-CrPV IRES-FLuc mRNA were translated for 60 min at 30°C in 15 μl of reticulocyte lysate that had been preincubated for 10 min with increasing amounts (0.2–1.5 μg) of IF3. Samples were analyzed by SDS–PAGE and autoradiography. (D) Structure of the CrPV IRES (Jan and Sarnow, 2002) showing the three pseudoknots, and toe-prints (black arrows) due to binding of 40S subunits (Wilson et al, 2000). mRNA triplets located in E, P and A sites are indicated.