CARP, INVOLVED IN PYRIMIDINE REGULATION OF THE ESCHERICHIA-COLI CARBAMOYLPHOSPHATE SYNTHETASE OPERON ENCODES A SEQUENCE-SPECIFIC DNA-BINDING PROTEIN IDENTICAL TO XERB AND PEPA, ALSO REQUIRED FOR RESOLUTION OF COLEI MULTIMERS

The carP gene involved in pyrimidine-specific regulation of the upstre am P1 promoter of the Escherichia coli carAB operon has been cloned in vivo on a mini-Mu replicon, sequenced and shown to be identical to th e xerB (pepA) gene encoding aminopeptidase A, a protein also involved in the Xer-mediated site-specific recombination at ColEI cer. The tran s-dominant allele carP6 was 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 a mino acid residues, was shown to be partially active; in the multicopy state the carPG allele can restore pyrimidine repressibility of the c arAB promoter P1. The trans-dominant character of the single copy carP G allele was found to be suppressed in the presence of multiple copies of the wild-type gene. The carP (pepA) control region was sequenced a nd transcription shown to be initiated at three promoters, the most up stream one of which was shown to be subject to negative autoregulation . The aminopeptidase activity of CarP (PepA) was found to be dispensab le for its role in pyrimidine-mediated repression of carAB transcripti on. CarP (PepA) was shown to be a sequence-specific DNA-binding protei n that does not require, at least not in vitro, any pyrimidine cofacto r to bind to the DNA. Mobility-shift and DNase I footprinting experime nts have revealed a specific binding of purified CarP (PepA) to two si tes in each one of the control regions of the E. coli and Salmonella t yphimurium carAB operons and to a single site in the carP (pepA) contr ol region. We propose that integration host factor and CarP/PepA-induc ed structural modifications in the carAB control region cause conforma tional changes required to assemble a pyrimidine-specific nucleo-prote in regulatory complex.