FIGURE 3.
Phosphotransfer from AtsR and its derivatives to the AtsT response regulator. Five μmol of AtsR, AtsRH245A, and AtsR-HK was preincubated in individual standard phosphorylation mixtures (100 mm Tris-HCl pH 8, 50 mm KCl, 5 mm MgCl2, 1 mm DTT, 5 μCi [γ-33P]ATP) for 15 min at 25 °C followed by the addition of 5 μmol of AtsT and/or BCAM0378. Reactions were terminated by adding 3× SDS-PAGE loading buffer after 15 min. Samples were resolved on 16% SDS-PAGE gel and stained with Coomassie Blue (bottom). Phosphorylated proteins were visualized by a PhosphorImager (Top). Phosphorylated bands corresponding to the expected masses of AtsR, AtsR-HK, and AtsT polypeptides are indicated by arrows.