FIG. 3.

Overexpression of TBP increases RNA Pol I-dependent promoter activity. (A) Overexpression of Drosophila TBP in S2 cells augments RNA Pol I-dependent promoter activity. S2 and TBP-S2 cells were transfected with 4 μg of pR119B as described in Materials and Methods and treated with CuSO₄ as indicated. RNA was extracted, RNase protection assays were performed with equal amounts of RNA, and the resultant labeled RNA digestion products were separated by gel electrophoresis and visualized by autoradiography. (B) Overexpression of human TBP in Huh7 cells augments RNA Pol I-dependent promoter activity. Huh7 cells were transiently cotransfected with increasing amounts of an expression plasmid containing a human TBP gene (pLTReTBP), as indicated, together with 4 μg of reporter plasmid phRR as described in Materials and Methods. RNase protection assays were performed with equal amounts of RNA from the transfected cells, and the resultant labeled RNA digestion products were separated by gel electrophoresis and visualized by autoradiography. (C) Inhibition of Ras activation does not block induction of RNA Pol I-dependent promoters in cells overexpressing TBP. S2 cells were transiently transfected with expression plasmids containing a ADH promoter-driven X cDNA (10 μg), an actin 5C promoter-driven TBP cDNA (0.5 μg), and an actin 5C promoter-driven dominant negative Ras (Ras-ala15) cDNA (2 μg), as shown, together with 4 μg of pR119B, as described in Materials and Methods. RNase protection assays were performed with equal amounts of RNA from the transfected cells, and the resultant labeled RNA digestion products were separated by gel electrophoresis and visualized by autoradiography.