Template opening in the presence
and absence of δTFIIS. Transcription
initiation complexes were assembled in the presence and absence
of δTFIIS on radiolabeled mutant AdML
templates as indicated at the top of the figure. (A)
Initiation complexes were assembled with AdML+23G (lanes
1–3), AdML+25G (lanes 4–6), AdML+26G
(lanes 7–9), AdML+27G (lanes 10–12) and
AdML+28G (lanes 13–15). The G residue creating
the stall site is indicated by the arrow. (B) Initiation
complexes were assembled with AdML+29G (lanes 1–3),
AdML+31G (lanes 4–6), AdML+33G (lanes
7–9), AdML+35G (lanes 10–12). The reactions included
nucleotides (60 µM ATP, 10 µM
UTP, 10 µM CTP and 120 µM
3′-OMeGTP) at 30°C
for 10 min. After this, potassium permanganate was added for 20 s
and the reactions were processed as described in Materials and Methods.
The last T residue before the stall site is indicated by the arrow
(see also Fig. 1).