Fig. 5. Binding of in vitro synthesized GAF to two putative GAGA binding sites at MCP residues 548 and 558. Lanes 1-5: EMSA performed in the absence of competitor. Oligonucleotide 55/56 contains the sequence between residues 533 and 572 that includes the putative GAGAG548 and GAGA558 binding sites (Fig. 7). Oligo 55/56M1 has the GAGA548 site mutated, oligo 55/56M2 has the GAGA558 site mutated, and oligo 55/56M3 has mutations in both sites. Radioactively labeled oligonucleotide 55/56 (lane 2) or one of the mutated versions (lanes 3-5) was incubated with in vitro-synthesized GAF factor and run on a 5% acrylamide gel (Materials and Methods). Two shifted bands (arrows) are present when incubated with GAF (lane 2) but not without (lane 1). The two shifted bands presumably correspond to protein-DNA complexes formed by the full-length and a shorter GAF polypeptides synthesized in our in vitro translation system (data not shown). Lanes 6-10: EMSA in the presence of competitors. The binding is competed by excess, unlabeled oligo 55/56 (lane 6) but not by non-specific competitor oligo (lane 10). Lanes 7-9 are competition experiments in which oligo 55/56 was labeled and the mutated oligos were used as excess, unlabeled competitors. The shifted band using 55/56M1 (lane 3) is faint. However, we believe this to be a true shift rather than due to spillover from adjacent lanes, as lane 7 shows shows that oligo 55/56M1 can compete for GAF binding.
