Figure 4.

CAD mutants that are defective for dTAF_II110 binding fail to recruit an RNA polymerase II complex to the promoter in vitro. (A) Wild-type CAD and the five mutant CAD proteins were synthesized without ³⁵S label by using the TNT system. Protein samples were run through a sephadex G-25 spin column to remove excess salts from the reactions. An aliquot of each purified TNT sample (15 μl) was tested by Western blotting with a primary antibody specific for the Gal-4 DNA binding domain to determine the amount synthesized. (B) Wild-type and mutant CAD proteins were tested for complex recruitment in an Ag-EMSA assay (see Materials and Methods). ³²P-labeled probe (5 fmol) was incubated with 3 μg of RLNE and 10 fmol of the appropriate purified proteins as indicated. The sample in lane 10 included primary antibody specific for the large subunit of RNA polymerase II, biotinylated secondary antibody, and streptavidin-coated beads to supershift the complex. Reactions were incubated at 4°C for 15 min and then separated on a 1% Seakem agarose gel. The gel was dried and exposed to film to visualize the complexes.