FIG. 2.

Effects of ICP4 and Gal4-VP16 on the formation of PICs. (A) Effect of ICP4 on PIC formation on immobilized HSV-1 gC promoter templates. Immobilized wt, TATA box-deleted (Δ TA), and INR-mutated (Δ INR) gC promoters (300 fmol) were incubated with HeLa nuclear extract (NE; 150 μg of total protein) in the absence or presence of purified ICP4 (300 ng) in a total volume of 75 μl for 90 min at 30°C. After incubation at 30°C, nucleoprotein complexes bound to immobilized promoters were isolated as described in Materials and Methods. The presence of ICP4, TFIIB, TFIID, and RNA Pol II was assessed by Western blot analysis. (B) Effect of Gal4-VP16 on PIC formation on immobilized G₅E4T promoter templates. Approximately 7,200 fmol of purified Gal4-VP16 (180 ng) and 300 fmol of immobilized G₅E4T were used as described for panel A but in a 300-μl volume and with direct elution of the promoter-bound proteins in SDS-PAGE sample buffer. The presence of Gal4-VP16, TFIIB, TFIID, and RNA Pol II was assessed by Western blot analysis.