FIG. 7.
Determination of the specific LexA binding target in the MC-1 umuDC promoter. PCR-directed mutagenesis was employed to change single nucleotides in the region from −48 to −31 (boldfaced) relative to the start codon of the MC-1 umuD gene (italicized). EMSA experiments allowed determination of the affinity of the purified LexA protein for the resulting DIG-labeled probes. Arrows point to the nucleotides used to replace the native nucleotides, and the relative positions of nucleotides with respect to the translation initiation codon are given in parentheses. The probe fragment with no change introduced is shown as a positive control (W). The percentage of LexA binding activity remaining after each change was assessed using ImageQuant 1.2 software (Binding). Key nucleotides mutations of which caused >50% decreases in LexA binding activity (compared to that of the wild-type control) are given at the bottom (Bases).