FIG. 5.
Effect of double mutations on transcription activation and binding by ComK. (A) Five single AT-box mutants were combined with an A-G substitution at position 2 in AT-box 2, the mutation seen in the wild-type comG promoter. β-Galactosidase (betagal) assays were performed to determine the effects of those mutations on transcription activation by ComK in vivo. The activities determined 4 h into the stationary growth phase are shown. β-Galactosidase activities and standard deviations are determined as described for Fig. 4, in this case relative to the activity in the wild-type-comG promoter K-box. (B) To determine the effect of the mutations on DNA binding by ComK, EMSAs were performed for different concentrations of ComK. Quantifications were performed as described for Fig. 4, and values are relative to that determined for the wild-type comG promoter K-box, used as a control (standard deviations are included for each mutant). Results are shown for the binding reaction with 0.07 μM ComK. Wt indicates the wild-type comG promoter K-box (the sequence of which is depicted above the graph in panel A; this K-box was used as the control in this experiment). With Ax or Tx, x indicates the position that is replaced by a G in the A or T stretch, respectively, of AT-box 1 (right AT-box), which is in addition to the A2-G substitution in AT-box 2 (left AT-box).