Two key proteins, LexA and RecA, are involved in regulation of the SOS expression system in bacteria. LexA and RecA act as the transcriptional repressor and inducer of the SOS operon, respectively. LexA downregulates the expression of at least 43 unlinked genes and activated RecA interacts with the repressor LexA and therefore, LexA undergoes self-cleavage. The ability of the LexA protein to dimerize is critical for its ability to repress SOS-regulated genes in vivo, as the N-terminal domain (NTD) alone has a lower DNA-binding affinity without the C-terminal domain (CTD) and the components for the dimerization of LexA are located in the CTD. Two antiparallel β-strands (termed β-11) in the CTD at the dimer interface of LexA are involved in the dimerization. LexA interacts with the active form of RecA in vivo during the SOS response. It was determined experimentally that monomeric and non-cleavable LexA binds more tightly to RecA and is resistant to self-cleavage. Therefore, we reasoned that if we can produce such LexA mutants we would be able to stabilize the LexA and active RecA complex for crystallization. Therefore, in this experiment, we attempted to make a non-cleavable and predominantly monomeric LexA that interacts intimately with RecA. We produced four single mutations at the dimer interface of the non-cleavable and NTD-truncated mutant of LexA (∆68LexAK156A) in order to weaken the interactions at the interface. The predominant forms of LexA mutants and the affinities of interaction between the mutant LexA proteins and RecA were examined. ∆68LexAK156AR197P mutant was found as predominantly monomeric at a concentration of 33.3 μM both by gel filtration chromatography and dynamic light scattering (DLS) experiments. It also bound RecA more tightly than wild-type LexA. Another mutant, ∆68LexAK156AI196Y, was also found as predominantly monomeric at a concentration of 33.3 μM by DLS. Both these proteins were subjected to crystallization with wild-type RecA protein. We were able to produce some predominantly monomeric LexA with good binding affinity for RecA; however, we were unsuccessful in co-crystallization.

Two key proteins, LexA and RecA, are involved in regulation of the SOS expression system in bacteria. LexA and RecA act as the transcriptional repressor and inducer of the SOS operon, respectively. LexA downregulates the expression of at least 43 unlinked genes and activated RecA interacts with the repressor LexA and therefore, LexA undergoes self-cleavage. The ability of the LexA protein to dimerize is critical for its ability to repress SOS-regulated genes in vivo, as the N-terminal domain (NTD) alone has a lower DNA-binding affinity without the C-terminal domain (CTD) and the components for the dimerization of LexA are located in the CTD. Two antiparallel β-strands (termed β-11) in the CTD at the dimer interface of LexA are involved in the dimerization. LexA interacts with the active form of RecA in vivo during the SOS response. It was determined experimentally that monomeric and non-cleavable LexA binds more tightly to RecA and is resistant to self-cleavage. Therefore, we reasoned that if we can produce such LexA mutants we would be able to stabilize the LexA and active RecA complex for crystallization. Therefore, in this experiment, we attempted to make a non-cleavable and predominantly monomeric LexA that interacts intimately with RecA. We produced four single mutations at the dimer interface of the non-cleavable and NTD-truncated mutant of LexA (∆68LexAK156A) in order to weaken the interactions at the interface. The predominant forms of LexA mutants and the affinities of interaction between the mutant LexA proteins and RecA were examined. ∆68LexAK156AR197P mutant was found as predominantly monomeric at a concentration of 33.3 μM both by gel filtration chromatography and dynamic light scattering (DLS) experiments. It also bound RecA more tightly than wild-type LexA. Another mutant, ∆68LexAK156AI196Y, was also found as predominantly monomeric at a concentration of 33.3 μM by DLS. Both these proteins were subjected to crystallization with wild-type RecA protein. We were able to produce some predominantly monomeric LexA with good binding affinity for RecA; however, we were unsuccessful in co-crystallization.