Figure 7.

Transcription factors required for the efficient transcription from the TBP-DR2-dependent promoter. (A) Transcription of pUhU6_0.35 and pUP_DR2 in vitro in the presence of TFIIIBα or TFIIIBβ. Increasing amounts of a TFIIIBα fraction (lanes 2 and 9, 10 µl; lanes 3 and 10, 20 µl; lanes 4 and 11, 30 µl) or a TFIIIBβ fraction (lanes 5 and 12, 2.5 µl; lanes 6 and 13, 5 µl; lanes 7 and 14, 10 µl) were reconstituted with 15 µl phosphocellulose fraction C (lanes 1–14). The samples with pUhU6_0.35 (lanes 1–7) were supplemented with 20 ng recombinant TBPwt (lanes 1–7) while the samples with pUP_DR2 (lanes 8–14) contained 20 ng TBP-DR2. Control reactions without TFIIIB activity are shown in lanes 1 and 8 respectively. (B) Replacement of phosphocellulose fraction C by PBP, TFIIIC1 and TFIIIU in the in vitro transcription of pUP_DR2 and pUP_DR2/hU6. Individual components of the PCC fraction were added in different combinations as indicated above each lane: PBP, 2 µg MQ-fraction; TFIIIC₁, 0.4 µg ESF-fraction; TFIIIU, 1.8 µg ESF-fraction. The reactions were incubated and processed as described, using 1 µg of pUP_DR2 (lanes 1–7) or 1 µg pUP_DR2/hU6 (lanes 8–14) as template. All reactions were supplemented with 20 ng recombinant TBP-DR2 and 20 µl TFIIIBα fraction. Lanes 1 and 8 show the positive control with 15 µl PCC fraction containing the TFIIIC1, pol III, PBP and TFIIIU activity. All other samples (lanes 2–7 and 9–14) contained 2 µl purified pol III.