Figure 4

Mutational analysis of one of the IpsA binding sites in the ino1 promoter. The importance of the predicted DNA sequence motif for IpsA binding was tested in electrophoretic mobility shift assays (EMSAs) with DNA fragments in which three nucleotides of the proposed motif were exchanged, as indicated. A + indicates that the mutated fragment was bound with the same affinity as the unaltered wild-type fragment (positive control); (+) indicates that the mutated fragment was shifted, but with lower affinity; - indicates that the mutated fragment was not shifted. In the case of M3 and M4, binding was completely abolished by the mutation, indicating that the six central base pairs are crucial for IpsA binding. For M1, M2 and M6 a slight decrease of binding was observed. The binding of M5 was unchanged.