FIG. 2.

CAST/hPAF49-specific antibodies repress rDNA promoter-directed Pol I transcription by UBF but have no effect on nonspecific Pol I transcription. (A) CAST/hPAF49-specific antibodies repress Pol I promoter-specific transcription. HeLa nuclear extracts (NE; 50 mM KCl) were incubated with CAST/hPAF49-specific antibodies (Ab; lane 1), buffer (lane 2), or preimmune sera (Preimm.; lane 3) for 30 min on ice. Immobilized template DNA (IT-rDNA) was added and, following incubation for a further 14 min, protein-IT-rDNA complexes were washed in TM10-0.05 M KCl. Transcription was initiated with the addition of NTPs, and the reaction was allowed to proceed for 30 min at 30°C. Transcript synthesis was analyzed by S1 nuclease protection assays and autoradiography. (B) CAST/hPAF49-specific antibodies do not affect nonspecific (promoter-independent, randomly initiated) transcription. HeLa nuclear extracts (NE; 50 mM KCl) were incubated with CAST/hPAF49-specific antibodies (Ab; lane 1), buffer (2), or preimmune (Preimm.) sera (3) for 30 min on ice. Nonspecific transcription was initiated by addition of sheared calf thymus DNA (ctDNA) as template and NTPs, and reactions were allowed to proceed for 45 min at 30°C. The efficiency of nonspecific transcription (transc) was determined in five independent experiments by measuring the radioactivity incorporated into the acid insoluble fraction and is expressed relative to that measured in the reaction with buffer alone, set at 100%. Standard deviations are indicated.