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 upstream 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 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 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 amino acid residues, was shown to be partially active; in the multicopy state the carP6 allele can restore pyrimidine repressibility of the carAB promoter P1. The trans-dominant character of the single copy carP6 allele 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 carAB transcription. CarP (PepA) was shown 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. coli and Salmonella typhimurium carAB operons 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 carAB control region cause conformational changes required to assemble a pyrimidine-specific nucleo-protein regulatory complex.
Charlier
D
D
Research Institute of the CERIA-COOVI, Brussels, Belgium.
Hassanzadeh
G
G
Kholti
A
A
Gigot
D
D
Piérard
A
A
Glansdorff
N
N
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D003086:Bacteriocin Plasmids; D001483:Base Sequence; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D043384:Glutamyl Aminopeptidase; D019426:Integrases; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011743:Pyrimidines / Q000378:metabolism; D045522:Recombinases; D011995:Recombination, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D017421:Sequence Analysis; D014158:Transcription, Genetic / Q000235:genetics
C087018:ArgR protein, Bacteria; C490177:ArgR protein, E coli; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011743:Pyrimidines; D045522:Recombinases; D012097:Repressor Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C115006:integron integrase IntI1; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)