Analysis of complex formation. (A) To determine the effect of sequence disruption on formation of the complexes, EMSA analyses were performed using three new oligonucleotide probes in which three hexanucleotide sequences, −462 to −457 (probe GM-2), −456 to −451 (probe GM-1), and −450 to −445 (probe GM-3), were mutated by changing the purines to pyrimidines and the pyrimidines to purines. (B) The results show that all three complexes, especially complex III, were unable to sufficiently bind when the sequence from −462 to −451 was mutated. (C) UV cross-linking analysis of complex III was performed to determine the specificity of its binding onto the G-455 probe and to approximate its molecular weight. All samples were binding reactions containing whole cell extract, were irradiated for 5 minutes (unless indicated otherwise), and were resolved on a 10% SDS-PAGE gel. Lane 1, no protein extract; lane 2, no irradiation; lane 3, protein extract (nonreduced); lane 4, protein extract (reduced); lane 5, addition of 100-fold unlabeled G-455 probe; lane 6, no extract, instead BSA (50 μg); lane 7, a probe with the same sequence as G-455 (Fig2A), but internally labeled on the bottom strand with [-³²P] dCTP, was used to verify the molecular weight of complex III, obtained from previous UV cross-linking analyses. After UV irradiation, DNAse I treatment, and isolation of the DNA-bound complex III band from a 4% nondenaturing gel, resolution on a 10% SDS-PAGE gel still shows that the approximate molecular weight of complex III is 47 kD. The results show that complex III is a protein of approximately 47 kD that specifically binds to the G-455 probe, containing the sequence from −468 to −439 of the Bβ chain gene, flanking the −455 nucleotide.