Identification of the Rhodobacter sphaeroides SOS box.

Abstract

Gel-mobility shift assays with crude cell extracts of Rhodobacter sphaeroides, which belongs to the alpha group of the proteobacteria, have shown that a protein binds to the promoter of its recA gene, resulting in two retardation bands. Analysis of the minimal region of the R. sphaeroides recA gene required for the formation of the DNA-protein complexes, revealed the presence of the motifs GTTCN7GATC and GAACN7GAAC, which are centred at positions -21 and +8 from the transcriptional starting point respectively. Using PCR mutagenesis, we have demonstrated that these two motifs are required for the formation of both DNA-protein complexes in vitro as well as for the DNA damage-mediated inducibility of the recA gene in vivo. Furthermore, the level of the recA gene expression in the constitutive mutants is the same as that achieved by the wild-type cells after DNA damage, indicating that the binding protein must be a repressor. The motif GTTCN7GTTC is also present upstream of the R. sphaeroides uvrA promoter, which in vitro specifically binds to a protein and whose expression is DNA damage inducible. Mutagenesis of this motif abolishes both the binding of this protein to the uvrA promoter and the DNA damage-mediated expression of this gene. The fact that the recA and uvrA wild-type promoters compete with each other for the retardation band formation, but not with their mutant derivatives in any of these motifs, indicates that the same repressor binds to the operator of both genes. All these results lead us to propose the sequence GTTCN7GTTC as the SOS box of R. sphaeroides. This is the first SOS box known whose sequence is a direct repeat and not a palindrome.