FIG. 1.

Activators target different components of the PIC for recruitment. (a) Overexpression of TBP inhibits the activity of promoter-bound TBP. When TBP is overexpressed with a reporter that contains Gal4 binding sites and a TATA box, transcription is activated. However, activation of the same promoter by Gal4-TBP is inhibited by overexpression of TBP. Gal4-TBP activity is unaffected by overexpression of LexA-VP16Δ456, even though LexA-VP16Δ456 squelches Gal4-VP16Δ456. (b) Cells were transfected in exactly the same manner as for panel a, and primer extensions for CAT and GAPDH mRNA were performed in parallel for each sample. The extension products were of the expected sizes. Numbers at left show sizes of DNA standards. (c) Overexpression of TBP does not affect the expression of Gal4-TBP protein, and expression of LexA-VP16Δ456 does not affect the expression of Gal4-VP16Δ456 protein when assessed by immunoblotting. (d) One possible model for the mechanism of inhibition of Gal4-TBP activity by TBP overexpression (see text for details). (e) Overexpression of TBP inhibits the activity of Gal4-Sp1 but potentiates the activity of Gal4-VP16Δ456. (f) Cells were transfected in exactly the same manner as for panel e, and primer extensions for CAT and GAPDH mRNA were performed in parallel for each sample. The extension products were of the expected sizes. The contrast of the image was increased to facilitate the visualization of the CAT mRNA in the cells transfected with the Gal4-Sp1 expression vector and the cells cotransfected with the Gal4-Sp1 and TBP expression vector. Numbers at left show sizes of DNA standards. (g) Overexpression of TBP does not affect the expression of the Gal4-Sp1 or Gal4-VP16Δ456 proteins when assessed by immunoblotting. (h) One possible model for the mechanisms of transcriptional inhibition and potentiation by TBP overexpression (see text for details).