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. 2005 Sep;73(9):5743–5753. doi: 10.1128/IAI.73.9.5743-5753.2005

FIG. 4.

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.

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