Figure 4.

Mutation of the IIARE in natural promoters reduces TFIIA binding to DNA and promoter activity. A, the DNA sequences for the natural core promoters and their derivatives showing the TATA box (bold), the IIARE consensus bases (blue), and the mutated IIARE consensus bases (red). B, mutation of the IIARE reduces TFIIA binding to the hCYP1A2 promoter. C, in vitro transcription assays showing the relative activity for the promoters of hCYP1A2–WT and hCYPEA2–mIIARE in the presence or absence of GAL4–VP16. D, luciferase assays showing the effect of mutation of the IIARE on the expression of a reporter gene driven by the human CYP1A2 gene promoter or its derivatives. E, immuoblotting of the samples to confirm the co-expression of Gal4–VP16 used in C. F, luciferase assays showing the effect of IIARE mutation on the expression of a reporter gene driven by the human FDPS1 gene promoter. G, immuoblotting of the samples with the co-expression of Gal4–VP16 used in E. Protein–DNA binding assays, in vitro transcription assays, luciferase assays, and Western blotting were performed as described in the legend to Fig. 3. Each column in C, D, and F represents the mean ± S.E. of three independent experiments. Significant difference was analyzed by comparing the activity of the IIARE-containing promoter with that of the IIARE-defect promoter. *, p < 0.05; **, p < 0.01. p value was obtained by Student's t test.