FIG. 6.

EMSA analysis of the effect of Rep78 on the interaction of TBP and the 16TATA oligonucleotide in the presence of Sp1. EMSA was performed as described for Fig. 5B except that 0.5× TBE running buffer was used. In assay A, GST-Rep78 and Sp1 (Promega Corporation, Madison, Wis.) were added to preformed TBP-16TATA complex individually or mixed (lanes 3 to 5). In assay B, TBP was incubated with GST-Rep78 and Sp1 separately or together prior to the addition of ³²P-labeled 16TATA oligonucleotide (lanes 8 to 10). Lane 1, free ³²P-labeled 16TATA oligonucleotide; lanes 2 and 7, TBP-16TATA complex; lane 3, addition of GST-Rep78 to the TBP-16TATA complex; lane 4, addition of Sp1 to the TBP-16TATA complex; lane 5, addition of a mixture of GST-Rep78 and Sp1 to the TBP-16TATA complex; lane 6, the Sp1-16TATA complex; lane 8, incubation of TBP with GST-Rep78 prior to the addition of 16TATA oligonucleotide; lane 9, incubation of TBP and Sp1 prior to addition of the 16TATA oligonucleotide; lane 10, incubation of TBP with a mixture of GST-Rep78 and Sp1 before the addition of the 16TATA oligonucleotide; lane 11, incubation of GST-Rep78 with 16TATA probe as a negative control. “Probe” indicates a free ³²P-labeled 16TATA oligonucleotide. I, II, and III, TBP-16TATA complex; fpu, footprinting unit.