FIG. 4.

Stability of Pol I PICs at the ribosomal promoter. (A) In parallel, for transcription and immunoblotting assays, PICs were assembled for 20 min at 4°C in nuclear extracts (NEs) (2 and 40 μl, respectively, and final volumes of 20 and 160 μl, respectively) with immobilized ribosomal promoter templates (IT-DNA, 2.5 and 20 μl of 50 ng of Fr4 DNA per μl of beads, respectively) as outlined. The immobilized DNA-Pol I transcription complexes were then washed at 50 mM KCl in TM10i buffer before being subjected to washes in the same buffer but with increased salt concentrations (50 to 200 mM KCl). (B) For immunobloting, proteins were eluted from the IT-DNA with 5 M urea, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted onto polyvinylidene difluoride membranes which were probed with antibodies against A190, TAF_I110, and UBF (lanes 1 to 3). (C) The parallel reactions were assayed for transcriptional activity, and transcripts were detected by S1 nuclease protection (lanes 1 to 3). In addition, to test for transcriptional recovery of the templates that had been washed at 100 and 200 mM KCl, 1 μl of purified Pol I was added back to the transcription reaction mixtures (lanes 4 and 5, respectively).