FIGURE 7.
9HE interferes with preinitiation complex assembly by affecting SL1 interactions with the rRNA promoter. A, the effect of 9HE on formation of Pol-I PICs was analyzed when the drug was added before (I), during (II), or after (III) PIC formation. Immobilized ribosomal promoter templates (IT) were initially incubated either with 10 μm 9HE (I) or with 10 μm 9HE and purified Pol-I, UBF, and SL1 (TF) (II) or with Pol-I factors only (III) for 15 min on ice. The beads were washed and incubated for an additional 15 min on ice either with Pol-I factors (I) or with buffer (II) or with 10 μm 9HE (III). The beads were washed and split in half and used either for in vitro transcription assay or for Western blot (WB) analysis. B, Pol-I PICs were assayed for transcriptional activity, and transcripts were detected by S1 nuclease protection and quantified. The data expressed as a percentage of the highest value (set at 100%) represent an average from three independent experiments. S.D. is shown. C, for immunoblotting, proteins were eluted from the IT-DNA with 8 m urea, subjected to SDS-polyacrylamide gel electrophoresis, and blotted onto PVDF membranes that were probed with antibodies against A135, PAF53, UBF, TAFI110, TAFI63, and TBP. D, the effect of 9HE on SL1 binding to the ribosomal promoter was analyzed when the drug was added either after (I) or during (II) binding. Immobilized ribosomal promoter templates were initially incubated either with purified SL1 (I) or with various amounts of 9HE (as indicated) and purified SL1 (II) for 15 min on ice. The beads were washed and incubated for an additional 15 min on ice either with various amounts of 9HE (I) or with buffer (II). The beads were washed and used for Western blot analysis. E, for immunoblotting, proteins were eluted from the IT-DNA with 8 m urea, subjected to SDS-polyacrylamide gel electrophoresis, and blotted onto PVDF membranes that were probed with antibodies against SL1 subunits TAFI110 and TAFI63.