FIGURE 8.

9HE inhibits multi- and single- round transcription with similar efficiency when added before or after PIC formation. A, the effect of 9HE on multi-round transcription was analyzed when various concentrations of the drug were added either before or after PIC formation. 2 μl of HeLa NE and 200 ng of supercoiled plasmid DNA template were incubated for 15 min on ice either with (before) or without (after) 9HE. 9HE was added to “after” reactions, and transcription was initiated by the addition of NTP. B, transcripts were analyzed by S1 nuclease protection, and a representative image is shown. C, the results were quantified with the aid of phosphorimaging. The data are expressed as a percentage of the highest value (set at 100%) and represent an average from three independent experiments. S.D. is shown. D, the effect of 9HE on single-round transcription was analyzed when various concentrations of the drug were added either before or after PIC formation. 4 μl of purified Pol-I, 1 μl of purified SL1, 0.1 μl of recombinant UBF, and 200 ng of supercoiled plasmid DNA template were incubated for 15 min on ice either with (before) or without (after) 9HE. 9HE was added to “after” reactions, and transcription was initiated by the addition of NTP. E, transcripts were analyzed by S1 nuclease protection, and a representative image is shown. The black triangle indicates a specific signal, and an asterisk indicates a nonspecific band. F, the results were quantified with the aid of phosphorimaging. The data are expressed as a percentage of the highest value (set at 100%) and represent an average from three independent experiments. S.D. is shown.