Figure 4.

Effect of TFIIS on recovery of RNA polymerase II in DNA-bound fraction. Free and DNA-bound RNA polymerase II complexes were separated as described, except that the whole-cell extract (50 μl, 20–30 mg protein/ml) was treated with TFIIS (gift from A. M. Edwards, McMaster University, Hamilton, Ontario, Canada) in the amounts indicated in a volume of 100 μl containing 50 mM Tris⋅Cl, pH 7.5/60 mM ammonium sulfate/10 mM magnesium chloride/10% glycerol/10 μM zinc sulfate for 30 min at 30°C, followed by chilling in ice, adjustment of potassium acetate concentration, and PEI precipitation. The amount of Rpb1 in the PEI pellet was determined by SDS/PAGE (10% gel) and immunoblotting. Phosphorylated and unphosphorylated forms of Rpb1 were not resolved in this percentage gel.