FIG. 5.

TFIIA is not required for efficient RNA polymerase III snRNA transcription from the U6 promoter in vitro. (A) Parallel in vitro transcription reactions from the Ad2ML and U6 snRNA promoters. Whole-cell extract (WCE) was supplemented with TBP (lane 1) and then treated with increasing amounts of control (nonimmune) antibody beads (lanes 2 to 6) or α-IIA antibody beads directed against the α and β subunits of TFIIA (lanes 7 to 11) at beads-to-extract ratios of 0.5:1, 1:1, 1.5:1, 2:1, and 2.5:1, respectively. (B) Immunoblot analysis of the extracts used in the experiment whose results are shown in panel A with the antibody directed against the α and β subunits of TFIIA. WCE plus TBP (4 μl) was loaded in lane 1, and the depleted extracts (6 μl) were loaded in lanes 2 to 11. The location of the α subunit is indicated at left. (C) Addition of TFIIA to TFIIA-depleted extracts does not stimulate U6 transcription in vitro in the presence of limiting amounts of recombinant TBP. Nuclear extract (NE) was treated with control (nonimmune) (lane 2) or anti-TFIIA (lanes 3 to 18) antibody beads at a 1:1 beads-to-extract ratio. E. coli-expressed TFIIA (2 μl) and TBP (amounts are indicated in nanograms) were added to the reaction mixtures as indicated above the lanes (lanes 4 to 18). The signals corresponding to correct transcription initiation were quantitated with a phosphorimager, the background was subtracted, and the numbers were normalized for the signal obtained in lane 3, which was set at 1. Ctr., control.