FIGURE 10.

9HE binds to SL1 but not to other components of the Pol-I machinery. A, HTETOP cells were incubated for 48 h with 1 μg/ml (+Tet) or without (no Tet) tetracycline and treated with low 9HE concentrations (0.1 μm) for various times as indicated. The relative level of pre-rRNA was determined by S1 nuclease protection assay. The results were quantified with aid of a phosphorimaging and are expressed as a percentage of the highest value (set at 100%) The data represent an average from three independent experiments, S.D. and statistical significance (***, p < 0.001) are shown. B, nuclear extract of cells transiently expressing FLAG-tagged components of the Pol-I transcription machinery (RRN3, Pol-I, SL1) or purified recombinant FLAG-UBF were incubated with α-FLAG beads for 2 h on ice and washed with TM10, 0.15 m KCl buffer, and beads were split in half. One-half was incubated with 10 μm 9HE; the other with buffer for 30 min on ice. After washing, proteins were eluted by FLAG-peptide, and activity of specific factor was assayed in a reconstituted transcription reaction. C, activity of immunopurified Pol-I factors, which were preincubated with or without 9HE, was measured by run-off assay. Top panel: Pol-I (lanes 5 and 6), RRN3 (lanes 7 and 8), and SL1 (lanes 9 and 10). Bottom panel: UBF (lanes 1–6). IP, immunoprecipitation. Transcription reactions in lanes 3–8 (top panel) were supplemented with purified SL1 and UBF, lanes 9–12 (top panel) were supplemented with Pol-Iβ and UBF, and lanes 1–9 (bottom panel) were supplemented with purified Pol-I and SL1. D, transcripts from C were quantified with 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. and statistical significance (***, p < 0.001; *, p < 0.05) are shown. p values have been calculated using one and two-way analysis of variance on R software.