Isolation and characterization of noncleavable (Ind-) mutants of the LexA repressor of Escherichia coli K-12.
The LexA repressor of Escherichia coli represses a set of genes that are expressed in the response to DNA damage. After inducing treatments, the repressor is inactivated in vivo by a specific cleavage reaction which requires an activated form of RecA protein. In vitro, specific cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH. We have isolated and characterized a set of lexA mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function. Forty-six independent mutants, generated by hydroxylamine and formic acid mutagenesis, were isolated by a screen involving the use of operon fusions. DNA sequence analysis identified 20 different mutations. In a recA mutant, all but four of the mutant proteins functioned as repressor as well as wild-type LexA. In a strain carrying a constitutively active recA allele, recA730, all the mutant proteins repressed a sulA::lacZ fusion more efficiently than the wild-type repressor, presumably because they were cleaved poorly or not at all by the activated RecA protein. These 20 mutations resulted in amino acid substitutions in 12 positions, most of which are conserved between LexA and four other cleavable proteins. All the mutations were located in the hinge region or C-terminal domain of the protein, portions of LexA previously implicated in the specific cleavage reactions. Furthermore, these mutations were clustered in three regions, around the cleavage site (Ala-84-Gly-85) and in blocks of conserved amino acids around two residues, Ser-119 and Lys-156, which are believed essential for the cleavage reactions. These three regions of the protein thus appear to play important roles in the cleavage reaction.
Lin
L L
LL
Department of Biochemistry, University of Arizona, Tucson 85721.
Little
J W
JW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D016685:Mitomycin; D008937:Mitomycins / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D012697:Serine Endopeptidases; D014157:Transcription Factors / Q000235:genetics*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial; D016685:Mitomycin; D008937:Mitomycins; D011938:Rec A Recombinases; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein