Figure 6

Identification of the SugR binding motifs by competitve EMSA studies. A) Detailed view of the ptsI/cg2118 intergenic region. For EMSA studies 15 pmol of the DNA fragments I2, I3, and I4 were used with 0.05 pmol of SugR protein. The DNA fragment I3 showed no binding of SugR, confining fragment A and fragment B carrying one SugR binding motif each. Similar sequences representing putative binding motifs in the fragments A and B are highlighted in bold letters. B) Validation of the binding motifs by competitive EMSA experiments. OligoA and oligoB represent doublestranded(ds)-oligonucleotides carrying the similar sequences of fragment A and B, respectively, with native bordering sequences. OligoAt and oligoBt represent those with modified bordering sequences. All four ds-oligonucleotides were used as competetive DNA fragments in EMSA experiments using 15 pmol purified SugR protein and 0.05 pmol of labeled DNA fragment I2. The I2/SugR-mixture was incubated with indicated amounts of the competitory (ds)-oligonucleotides. The mixture of the DNA fragment I2 and the SugR protein was used as positive control and the DNA fragment I2 alone as negative control.