Figure 4.

Transient cotransfection assay. Human 293 cells were cotransfected, using the calcium phosphate precipitation method with (i) 1 μg of expression plasmid encoding yTBP or TBP/ZF, (ii) 5 μg of activator plasmid, GAL4-VP16, (iii) 0.5 μg of β-galactosidase expression plasmid (pCMVβ) as an internal control, (iv) 1 μg of a reporter plasmid (derived from pGL3-Basic) encoding the firefly luciferase gene, and (v) variable amount of the carrier plasmid (pUC19) to keep the total amount of transfected DNA at 20 μg. Each reporter construct had five GAL4-binding sites upstream of one of the promoter sequences (TATA, TATA/CGC, or GCG/TATA) used in the in vitro transcription assay (Fig. 3). In a parallel assay of basal transcription, GAL4-VP16 was omitted. Luciferase activity was measured 2 days after transfection and was normalized (i) with respect to β-galactosidase activity (to correct for transfection efficiency), and (ii) to the corresponding value from the cells transfected with blank expression vector, pcDNA3 (which was set to an arbitrary value of 10⁴). The absence or presence of GAL4-VP16 is indicated. The data represent an average of three independent experiments, and the standard error of the mean is shown.