FIG. 4.

Hydroxyl radical protection of TFIIIB-TBP-DNA complexes. (A) Summary of hydroxyl radical protection at U6-MLP TATA by TFIIIB. (B) Hydroxyl radical footprinting of the U6-MLP. The panel on the far right shows hydroxyl radical footprinting of the bottom strand of the U6-MLP, using either of the two C-BRF derivatives. TFIIIB indicates that TBPc, BRF, and B" were all added. The intensity of each base in each reaction was quantitated and normalized to the corresponding intensity in unbound DNA. Those bases that are reproducibly protected with the addition of TBPc, BRF, and B" are bracketed. Protection by TFIIIB was defined as a greater than 25% decrease in intensity compared with TBPc bound alone. Hydroxyl radical protection of the U6-MLP TATA nontranscribed strand with TFIIIB containing either C-BRF derivative shows similar protection to that of TFIIIB containing wild-type BRF (10a).