Figure 3.

In vitro transcription analysis. Biotinylated DNA fragments containing the promoters (TATA, TATA/CGC, and GCG/TATA) upstream of a G-free cassette were immobilized on streptavidin-coupled paramagnetic beads and used as transcription templates. yTBP at 6 μg/ml (lanes 1–3 and 7–9) or TBP/ZF at 8 μg/ml (lanes 4–6 and 10–12) was preincubated with each template (0.1 nM) for 1 h at room temperature. Then, supernatants were removed, and excess amounts (1 μM each) of competitor DNA oligonucleotides (GCG and TATA from Fig. 2A) were added to the preincubation mixture. After incubating 24 h at 4°C, the beads were washed to remove proteins that dissociated from the templates, and human transcription factors (TFIIB, -IIE, -IIF, and -IIH), RNA polymerase II, and substrate nucleotides were added to initiate transcription. yTBP was also added to a final concentration of 0.2 μg/ml in lanes 7–12. The transcripts were analyzed by urea gel electrophoresis.