Fig 7.

In vivo interactions between AD1+2 and TBP analyzed by the transcriptional interference assays. (A) Schematic shows full-length and truncated T7-tagged TBP proteins coexpressed with GAL4-AD1+2. (B) Western blot of T7-tagged TBP mutants transiently expressed in HeLa cells and detected with anti-T7-tag antibody. Numbers indicate constructs depicted in panel A. (C) Transcriptional interference assay demonstrates the effect of coexpressed TBP mutants on the transcriptional activity of AD1+2. One effector plasmid contained HSF1 activation domains AD1+2 fused to the GAL4 DBD in vector pNG-SB. The other effector consisted of the indicated TBP deletions with the T7-tag and nuclear localization signal cloned into vector pT7-NLS. The reporter was the luciferase gene with 5 GAL4 DNA-binding sites upstream of the TATA-box (pG5luc). Lane marked “C” contained pCI-neo as an empty-vector control with only polylinker downstream of the CMV promoter. The following DNAs were cotransfected into HeLa cells: 0.3 μg of GAL4-AD1+2, 3 μg of the TBP mutant, 2 μg of pG5luc, and 0.5 μg of the growth hormone reporter (pXGH5) as an internal standard. For all assays, luciferase activities of triplicate transfections were normalized against production of human growth hormone to establish relative activities. Activity of pCI-neo alone was assigned a value of 100%. (D) Transcriptional interference assay demonstrating little effect of TBP mutants on the transcriptional activity of VP16. VP16 activity was monitored using a mammalian 2-hybrid system to reconstitute VP16 activity by coexpression of GAL4 DBD-P and N-VP16 vectors (Patel et al 1995). Coexpression of TBP mutants and the empty-vector control were as described in panel C. The ratio of VP16 effector to TBP mutant DNAs was 1:10 in transfections. Activity of the VP16 effector alone was assigned a value of 100%. Activity of GAL4-HSFAD1+2 was 0.39 times the activity of the 2-hybrid GAL4-VP16