Figure 2.

N augments the translational expression of capped mRNA. We examined the effect of increasing N on the translational expression of three reporter mRNAs containing or lacking a 5′ cap using rabbit reticulocyte lysates. Capped and uncapped mRNA encoding GFP, luc or N were translated in vitro in the presence of ³⁵S-methionine and increasing amounts of N in (A–C), respectively. Translation products were then electrophoresed on SDS polyacrylamide gels, and the amount of translation product was quantified by phosphorimage analysis. Translation of capped and uncapped RNA is depicted with filled and open squares, respectively. The amount of labelled protein synthesized with capped RNA in reactions lacking N was normalized to 1 (this cannot be indicated on the log scale). In the absence of N, expression of the indicator proteins was slightly higher with uncapped than capped RNA. This is consistent with earlier observations (Svitkin et al, 1996). Thus, the amount of expression at the lower concentrations of N is equivalent to background levels of expression from capped and uncapped RNA for each of the indicator RNAs.