The nucleotide sequence and characterization of the relA gene of Escherichia coli

The relA gene product of Escherichia coli is known to be responsible for the synthesis of guanosine 3',5'-bispyrophosphate (ppGpp) during the stringent response to amino acid starvation. This report presents the sequence of the relA gene region and assignment of its 743-codon open reading frame by the following criteria: 1) genetic complementation of ppGpp synthesis in a relaxed (relA1) mutant during the stringent response; 2) changes in 3-aminotriazole resistance during growth to mimic a relA+ phenotype; 3) verification of the presence of an amber codon at the normal carboxyl terminus of the relA gene; and 4) immunological assays of expression of the RelA protein. The apparent molecular mass of the cloned relA gene product is calculated to be 83,856 daltons and as visualized by immunoblotting is identical to that of the previously characterized protein. A promoter has been identified that directs relA gene transcription towards the pyrG gene, in a counterclockwise direction on the E. coli chromosome. Genomic Southern blot analyses verify that the relA regions cloned and subjected to nucleotide sequence analysis correspond to homologous regions on the E. coli chromosome.