FIG. 9.

TFIIE stimulates transcription from the RNA polymerase II snRNA U1 promoter. (A) Addition of E. coli-expressed TFIIE to TFIIE-depleted extract. Nuclear extract (NE) was treated with control (nonimmune) antibody beads (lane 2) or anti-TFIIE antibody beads directed against the p34 subunit of TFIIE (α-IIEp34; lanes 3 to 6) at a 2:1 beads-to-extract ratio and used in transcription reactions with linearized Ad2ML and U1 templates. In lanes 4 to 6, 0.5, 1.5, and 3 μl of E. coli-expressed TFIIE was added to the reaction mixtures. (B) Immunoblot analysis of the extracts used to obtain the results shown in panel A with antibodies directed against the p34 and p56 subunits of TFIIE. (C) Addition of GTFs to TFIIE-depleted extracts. Nuclear extract was treated with control (nonimmune) antibody beads (lane 2) or α-TFIIEp34 antibody beads (lanes 3 to 10) at a 1:1 beads-to-extract ratio and used in parallel transcription reactions with linearized Ad2ML and U1 templates. In the reaction whose results are shown in lane 4, a combination of recombinant TFIIE (1 μl), purified RNA polymerase II (pol II; 1.5 μl), recombinant TFIIA (1 μl), recombinant TFIIB (2 μg), recombinant TFIIF (1 μl), and purified TFIIH (2 μl) was added. In lanes 5 to 10, the same combination of GTFs was added except for the factor indicated above each lane. (D) Immunoblot analysis of the extracts used to obtain the results shown in panel C with antibodies directed against the p34 and p56 subunits of TFIIE, the largest subunit of RNA polymerase II, and TFIIB. Ten microliters of extract was loaded per lane. Ctr., control.