FIG. 6.
Heparin resistance of preinitiation complexes containing wild-type or mutant TFIIIC. Wild-type or mutant TFIIIC were preincubated for 10 min at 25 or 40°C as indicated. Biotin-coupled SUP4 tRNATyr gene was then reacted with streptavidin beads (Dynabeads), and preinitiation complexes were formed by addition of rBrf1, rTBP, and rB" onto immobilized DNA. After 60 min at 25°C, heparin (20 or 50 μg/ml) was added and incubation was pursued for 5 min. Beads were then washed to remove TFIIIC, and single-round transcription on SUP4 tDNATyr was performed as described in Materials and Methods. The products were run on a 7 M urea, 6% polyacrylamide gel and revealed by autoradiography. The signal obtained for the mutant factor (left) is weaker than that obtained with the wild-type factor (right), as less protein was used due to a salt concentration limitation.