Figure 3

Binding of TIPT2 and/or TBP to TATA box-containing promoters. (A) Analysis of GST-TIPT2 binding to AdMLP. The specificity of the DNA-binding activity of 30 ng GST-TIPT2 was verified by competition with cold poly(dG-dC), poly(dI-dC), and unlabeled (*) AdMLP oligonucleotides, respectively. (B) The specificity of TBP and TIPT2 binding to AdMLP was tested by antibodies competition. Lane 2: The binding of 6 ng TBP to AdMLP. Lane 3: TBP-AdMLP complex was competed by anti-TBP antibodies. Lane 4: The binding of 15 ng GST-TIPT2 to AdMLP oligonucleotides. Lane 5: TIPT2-AdMLP complex was competed by anti-GST antibodies. (C) The binding of human TBP (6 ng) or GST-TIPT2 (30 ng) to AdMLP (containing BRE elements and a TATA box), AdMLPm1 (TATA box mutated) and AdE4 (no BRE elements). For sequences see Figure 3E. (D) GST-TIPT2 binding to AdMLP and m2–m9 mutants. (E) Oligonucleotide sequences of AdMLP representing AdMLP (BRE elements in bold), nine mutations of AdMLP (mutated bases highlighted), and AdE4. The TATA box motifs are framed. (F) Competition of the interaction between AdMLP and TIPT2 by mutated versions of AdMLP. Unlabelled oligonucleotides were preincubated with 6 ng GST-TIPT2. For sequences see Figure 3E. (G) Simultaneous binding of TIPT2 (60 ng) and TBP (6 ng) to AdMLP. The data in Figure 3B, D, F, G, respectively, stem from the same gel and exposure, the assembly is indicated by white lines.