Fig 4. Construction and validation of recA promoters engineered with different LexA operators.

A. Site-directed mutagenesis of the recA promoter’s operator sequence to create 22 synthetic SOS promoters. Transcription start site is indicated by rightward facing arrow and conserved -10 and -35 RNAP binding sites are underlined. LexA operator sequence indicated by top bracket. Mutagenesis was restricted to the region indicated, except for base pair position 18 of the dinG operator. The first group of sixteen promoters was engineered to contain operator sequences which mimic the consensus operator and are identical to one another, except for operator base pair positions 14 and 15. The second group of six promoters was engineered to contain operator sequences found in E. coli SOS genes. Bolded residues indicate deviations from the consensus operator sequence. The ‘scram’ promoter contains an operator sequence in which the highly conserved CTG (CAG)-motifs of the LexA operator sequence (grey shading) were mutated to ablate LexA binding. Binding is not detectable to the scram operator in biochemical assays at the highest LexA concentration tested of 1 μM. B. LexA operator half-site alignment. LexA binds to its 20 bp operator DNA as a dimer. Operators are comprised of two half-sites, which exhibit dyad symmetry with respect to highly conserved CTG (CAG)-motifs. Each monomer of a LexA dimer engages one half-site of the operator. The DNA sequence alignment contains 60 half-sites, derived from 30 operators in the E. coli chromosome. Sequences are arranged as in Fig 3C, in order of increasing t_1/2 value. For each operator, the DNA sequence of the ‘right’ half-site is shown above the ‘left’ half-site. ‘Left’ half-site sequences are reverse complemented to account for the dyad symmetry. Residue frequencies for each position and the consensus half-site sequence are given below the alignment. The consensus frequency values are bolded, the highly conserved CTG (CAG)-motif is highlighted, and the residues targeted for mutagenesis in A (positions 14 and 15) are outlined with a black border. C. Basal promoter activity of the synthetic recA promoters as a function of LexA-operator dissociation rate (t_1/2) in ΔlexA, lexA⁺, and lexA^S119A cells. Horizontal dotted line indicates the value obtained with the ‘scram’ control promoter in the lexA⁺ strain.