Figure 3.

Depletion of hB′′ debilitates RNA polymerase III, but not RNA polymerase II, transcription in vitro. Whole-cell extract was treated with beads coated with either the preimmune antibodies (lanes 1 and 5) or the anti-B′′ antibodies (lanes 2–4 and 6–8) indicated above the lanes (913 is αhB′′-2; 843 is αhB′′-3). In lanes 3 and 7, and in lanes 4 and 8, the antibody beads were preincubated either with the specific peptide against which the antibodies were raised or with nonspecific peptide, as indicated. In lane 9, the extract was treated with beads coated with an antibody directed against the SNAP43 subunit of SNAP_c. Aliquots of the variously treated extracts were then tested in parallel for their ability to direct transcription from the Ad2 VAI, human U6, human U1, and Ad2 major late (AdML) promoters. The bands corresponding to correctly initiated RNA are indicated in each panel. In the U1 panel, the band labeled 5′ RT corresponds to RNA initiated within the vector and reading through the U1 snRNA promoter (Sadowski et al. 1993). Lane 10 shows transcription in untreated whole-cell extract.