FIG. 4.
MtsR binding to the sia promoter region is specific and metal dependent. A. Binding of the purified rMtsR to Pshr fragment. All EMSAs were done with the 32P-labeled promoter fragment. Additional components of the different binding reactions are indicated above the lanes. Lane 1 from the left, [32P]Pshr fragment only. Lane 2, [32P]Pshr fragment but with 7.5 ng of purified MtsR-His6. Lane 3, the same as in lane 2 but with 250 μM EDTA. Lane 4, the same as in lane 3 but with 25 μM FeSO4. Lane 5, the same as in lane 3 but with 75 μM FeSO4. Lane 6, the same as in lane 3 but with 50 μM FeSO4. Lane 7, the same as in lane 3 but with 100 μM FeSO4. Lane 8, the same as in lane 2 but with a 10-fold increase in the level of unlabeled (cold) Pshr fragment. Lane 9, the same as in lane 2 but with a 10-fold increase in the level of unlabeled recA fragment. Lane 10, [32P]Pshr fragment and 100 ng of SiaA-His6 (4). B. Iron restores binding of EDTA-treated rMtsR to DNA. Lane 1, [32P]Pshr fragment only. Lane 2, the same as in lane 1 but with 10 ng of rMtsR pretreated with EDTA. Lane 3, the same as in lane 1 but with 10 ng of untreated rMtsR. Lane 4 the same as in lane 2 but with 25 μM FeSO4. Lane 5, the same as in lane 2 but with 75 μM FeSO4. Lane 6, the same as in lane 2 but with 150 μM FeSO4. C. Manganese restores binding of EDTA-treated rMtsR to DNA. Lane 1, [32P]Pshr fragment only. Lane 2, the same as in lane 1 but with 10 ng of rMtsR pretreated with EDTA. Lanes 3 and 4, the same as in lane 2 but with 25 and 50 μM MnCl2, respectively.