Figure 2.

Displacement of mediator but not TFIIF from RNA polymerase II holoenzyme by anti-CTD antibodies. Holoenzyme (100 μg) was incubated with 8WG16 monoclonal antibodies immobilized on protein A–Sepharose beads in 20 mM Tris⋅acetate, pH 7.8/20% glycerol/200 mM potassium acetate/0.01% Nonidet P-40/0.1 mM EDTA, as described (13) for 4 h at 4°C. The beads were removed by sedimentation in a microcentrifuge for 5 min at 5,000 rpm, and starting material (holopol II) and supernatant proteins (mediator) were compared by SDS/PAGE and silver staining. The top portion of a 10% gel is shown. Protein components of the holoenzyme preparation are indicated on the left. In addition to the Tfg1 subunit of TFIIF, the two smaller Tfg2 and Tfg3 subunits failed to appear in the supernatant (mediator) fraction, as did the subunits of RNA polymerase II.