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, 33 (19), 6287-95

Binding of the Bacillus Subtilis LexA Protein to the SOS Operator

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Binding of the Bacillus Subtilis LexA Protein to the SOS Operator

Eli S Groban et al. Nucleic Acids Res.

Abstract

The Bacillus subtilis LexA protein represses the SOS response to DNA damage by binding as a dimer to the consensus operator sequence 5'-CGAACN(4)GTTCG-3'. To characterize the requirements for LexA binding to SOS operators, we determined the operator bases needed for site-specific binding as well as the LexA amino acids required for operator recognition. Using mobility shift assays to determine equilibrium constants for B.subtilis LexA binding to recA operator mutants, we found that several single base substitutions within the 14 bp recA operator sequence destabilized binding enough to abolish site-specific binding. Our results show that the AT base pairs at the third and fourth positions from the 5' end of a 7 bp half-site are essential and that the preferred binding site for a LexA dimer is 5'-CGAACATATGTTCG-3'. Binding studies with LexA mutants, in which the solvent accessible amino acid residues in the putative DNA binding domain were mutated, indicate that Arg-49 and His-46 are essential for binding and that Lys-53 and Ala-48 are also involved in operator recognition. Guided by our mutational analyses as well as hydroxyl radical footprinting studies of the dinC and recA operators we docked a computer model of B.subtilis LexA on the preferred operator sequence in silico. Our model suggests that binding by a LexA dimer involves bending of the DNA helix within the internal 4 bp of the operator.

Figures

Figure 1
Figure 1
Binding of B.subtilis LexA to SOS operators. Graphical analyses of mobility shift titrations of 32P-labelled recA (B), uvrB (C), dinB (D) and dinC (E) promoter DNA (5.0 nM) incubated with increasing concentrations of purified LexA (black circles) or crude extract (open circles). (A) A typical mobility shift assay with the recA promoter as described in Materials and Methods. (B–E) Typical plots of fractional saturation of promoter DNA versus the concentration of 'unbound LexA.
Figure 2
Figure 2
Inhibition of LexA binding to the recA operator by recA operator mutants. Mobility shift assays were conducted as described in Materials and Methods with purified LexA, radiolabeled recA promoter DNA (5.0 nM) and a 100-fold molar excess of competitor recA operator mutant. Base changes are indicated by position from the 5′ end of the 14 bp operator, and the base substitution relative to the recA operator sequence. Lower and upper bands correspond to unbound and LexA-bound recA promoter DNA, respectively. Lanes corresponding to no addition of competitor DNA and non-specific DNA [poly(dI–dC)] are indicated. LexA concentrations given are for the total amount of LexA.
Figure 3
Figure 3
Representative Scatchard plots for quantifying inhibition of LexA binding to the recA operator by operator mutants. Mobility shift assays were conducted as described in Materials and Methods with purified LexA, radiolabeled recA promoter DNA (5.0 nM), and a 100-fold molar excess of wild-type recA operator (black circles), recA operator with A substituted for C at the first position (black triangles) and no competitor (black squares). LexA concentrations used were for unbound LexA.
Figure 4
Figure 4
Sequence requirements for LexA binding. The preferred half-site sequence based on thermodynamic analysis of LexA binding to recA operator mutants. Base substitutions labeled as destabilizing abolish LexA binding to the recA operator.
Figure 5
Figure 5
Computer model of B.subtilis LexA superimposed on the structure of E.coli LexA. A homology model of the B.subtilis LexA protein (turquoise) was superimposed on the crystal structure of the E.coli LexA protein (purple) as described in Materials and Methods.
Figure 6
Figure 6
Computer model of the putative B.subtilis LexA DNA binding domain docked at one half-site of the preferred operator sequence CGAACATATGTTCG. The homology model depicted in Figure 5 was docked on the preferred operator sequence in silico as described in Materials and Methods.
Figure 7
Figure 7
Computer model of LexA dimer bound to the preferred operator sequence CGAACATATGTTCG. A second LexA monomer was docked on the preferred operator sequence in the same orientation as shown in Figure 6. Side view (A) and top view (B) of the model of the LexA dimer bound to DNA show that distortion of the DNA and/or LexA must occur for the C-terminal domains to interact effectively.

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References

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