Figure 4. Translational activity of the 5′-UTR. A: Structure of the expression vector and inserted sequence. The psiCHECK-2 PCR vector is described (bottom). This vector contains the Renilla (R) and firefly (F) luciferases as the upstream and downstream cistrons, respectively. Both the α5′-UTR and the β5′-UTR were amplified (upper and middle, respectively) and inserted into the cloning site. Before inserting the 5′-UTR sequences into the psiCHECK-2 vector, the ATG of the Renilla luciferase was mutated to TTG, so that the Renilla luciferase expression would be driven by the primary ATG initiation codon (reverse triangle) of the gene under investigation. B: Luciferase activity. Three vector constructs (the unmodified plasmid psiCHECK-2-TTG and plasmids psiCHECK-2-α and -β) were transfected into HEK293 cells, and the firefly and Renilla luciferase activities were measured 24 h after transfection. All experiments were performed in triplicate. The ratio of Renilla and firefly luciferase activity was normalized against the unmodified plasmid psiCHECK-2-TTG. The relative ratio of Renilla/firefly luciferase activity determined from cells transfected with the plasmid psiCHECK-2-α was set at 1. The relative ratio of Renilla/firefly luciferase activity was significantly lower for the β5′-UTR than for the α5′-UTR (0.71±0.01 and 1.00±0.01, respectively).