carP, Involved in Pyrimidine Regulation of the Escherichia coliCarbamoylphosphate Synthetase Operon Encodes a Sequence-specific DNA-binding Protein Identical to XerB and PepA, also Required for Resolution of ColEl Multimers

The carPgene involved in pyrimidine-specific regulation of the upstream P1 promoter of the Escherichia coli carABoperon has been cloned in vivoon a mini-Mu replicon, sequenced and shown to be identical to the xerB( pepA) gene encoding aminopeptidase A, a protein also involved in the Xer-mediated site-specific recombination at ColEI cer. The trans-dominant allele carP6was cloned as well and shown to bear a single G→A transition that converts the TGG codon (Trp473) into a TAG amber stop codon. The truncated mutant protein, missing the 31 C-terminal amino acid residues, was shown to be partially active; in the multicopy state the carP6allele can restore pyrimidine repressibility of the carABpromoter P1. The trans-dominant character of the single copy carP6allele was found to be suppressed in the presence of multiple copies of the wild-type gene. The carP( pepA) control region was sequenced and transcription shown to be initiated at three promoters, the most upstream one of which was shown to be subject to negative autoregulation. The aminopeptidase activity of CarP (PepA) was found to be dispensable for its role in pyrimidine-mediated repression of carABtranscription. CarP (PepA) was showed to be a sequence-specific DNA-binding protein that does not require, at least not in vitro, any pyrimidine cofactor to bind to the DNA. Mobility-shift and DNase I footprinting experiments have revealed a specific binding of purified CarP (PepA) to two sites in each one of the control regions of the E. coliand Salmonella typhimurium carABoperons and to a single site in the carP( pepA) control region. We propose that integration host factor and CarP/PepA-induced structural modifications in the carABcontrol region cause conformational changes required to assemble a pyrimidine-specific nucleo-protein regulatory complex.