NC2 inhibits basal transcription and stimulates activated transcription in vitro. (A) BUR6+ gal4Δ and bur6–1 gal4Δ strains transformed with either Gal4-VP16 or vector CEN plasmids were replica plated to SC-Leu glucose (Glu) or SC-Leu galactose (Gal) plates. As observed previously with GAL4, GAL4-VP16 is defective for activation in the bur6–1 strain. (B) Western blot detection of Bur6, Ydr1, and TBP levels in whole-cell extracts prepared from BUR6+ and bur6–1 strains. (C) In vitro transcription assays with wild-type (lanes 1 and 2) or bur6–1 (lanes 3–10) whole-cell extract with purified Gal4-VP16 and yNC2 proteins added as indicated at the top. Transcription levels are indicated at the bottom, relative to basal transcription from the bur6–1 extract. (D) Reactions were assembled as in C, and either 1, 4, or 16 ng of recombinant Bur6, Ydr1, or Bur6 + Ydr1 (NC2) were added as shown.