FIGURE 4.

Effect of added eIF4F and its individual subunits on translation in factor-depleted extracts. (A) Wge depleted of cap-binding complex (50 μL) (as in Fig. 3) was supplemented with indicated amounts of eIF4F or its subunits (eIF4E, eIF4G) and programmed with BLucB mRNA (20 nM). (B) Depleted wge (50 μL) was supplemented with indicated factors and programmed with BLucB, capped BLucBBF mRNA (cap-BLucBBF), and capped reporter RNA containing vector-derived UTRs and a 68-nt poly(A) tail [capVLucV(A)₆₈] (final concentration 20 nM). The level of translation was expressed as a percentage of relative light units (RLU) obtained from luciferase translated in each extract supplemented with eIF4F. In the presence of eIF4F (defined as 100% for each mRNA), luciferase expression for capVLucV(A)₆₈ was 3000 RLU, while capBLucBBF, and uncapped BLucB RNAs yielded between 5000 and 6000 RLU. (C) Depleted wge (50 μL) was supplemented with indicated factors and programmed with BLucB (20 nM). The 86-kDa truncation mutant of eIF4G, lacking the eIF4E recognition site is indicated as 4G86. The 1:1 mixture of eIF4E and 4G86 is indicated as 4F86. The level of translation is expressed as the percentage of luciferase produced in the extract supplemented with eIF4F. The data are averages of triplicates from at least two experiments, and error bars represent standard error.