Fig. 4.

Perturbing an in vitro transcription system with TBP-aptamers. A template with a TATA-dependent promoter (2.25 nM) was incubated with yeast whole cell extract (containing ≈20 nM TBP) for 30 min to allow PIC formation. The transcription reaction was started by addition of NTPs and allowed to proceed for 30 min, unless otherwise indicated. (A) Aptamers inhibiting transcription when present during PIC formation. The concentration indicated is that of the aptamers or TATA-DNA included in each reaction. (B) The effect of adding aptamers after PIC formation. Aptamers (160 nM) were added after 30-min incubation of templates with cell extract. To examine single-round transcription (Top), sarkosyl was added to 0.25% final concentration 30 s after transcription initiation. Multiple-round transcription experiments were allowed to proceed for the time indicated. The quantity indicated below each lane represents the percentage of that in the reaction that did not include aptamers.