Pyrimidine regulation of the Escherichia coli and Salmonella typhimurium carAB operons: CarP and integration host factor (IHF) modulate the methylation status of a GATC site present in the control region.
By measuring the protection against Dam methylase modification of a GATC sequence located 106 bp upstream of the startpoint of promoter P1 in the control region of the carAB operon (encoding carbamoylphosphate synthetase) we have obtained evidence for a direct correlation between the degree of in vivo occupancy of a specific regulatory target site and the repressibility of the P1 promoter by pyrimidine residues. A high uridine nucleotide pool as well as binding of the carP (alias xerB/pepA) gene product and of the integration host factor (IHF) to the carAB control region are prerequisites to observe this in vivo protection. Purified CarP binds in vitro to the carAB control region and protects against DNase I two approximately 25 bp long stretches, one of which is located just downstream of the GATC sequence. Mutations in this site strongly impair the pyrimidine regulation of the P1 promoter and the interference with Dam methylase modification. These processes are also strongly impaired in the absence of integration host factor and in mutants affected in the IHF site located some 200 bp upstream of this Dam methylase modification site. IHF therefore exerts at least part of its antagonistic effects on P1, i.e. increased expression in minimal medium but increased repression in the presence of pyrimidine residues, indirectly by influencing the formation or the stability of a particular protein-DNA complex. Furthermore, we demonstrate that the distance separating the IHF and Dam methylase target sites is crucial for the in vivo protection and for pyrimidine-mediated regulation of the promoter expression. Mutations altering this distance result in severe reductions of the degree of in vivo protection and, concomitantly, of the repressibility by pyrimidine residues of promoter P1 activity in a way indicative of the formation of a complex nucleoprotein structure. Since neither IHF nor CarP require pyrimidine residues to bind to the carAB control region, at least not in vitro, it is tempting to suggest that IHF and CarP-induced bending and looping provide changes in DNA topology that are required for assembling a specific pyrimidine-dependent nucleoprotein complex that modulates P1 activity.
Charlier
D
D
Research Institute of the CERIA-COOVI, Brussels, Belgium.
Gigot
D
D
Huysveld
N
N
Roovers
M
M
Piérard
A
A
Glansdorff
N
N
D000225:Adenine / Q000378:metabolism; D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D043384:Glutamyl Aminopeptidase; D034982:Integration Host Factors; D008745:Methylation; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic; D011743:Pyrimidines / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014500:Uracil Nucleotides / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D034982:Integration Host Factors; D011743:Pyrimidines; D012097:Repressor Proteins; D014500:Uracil Nucleotides; C440508:integration host factor, Salmonella; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499294:dam protein, E coli; D003850:Deoxyribonuclease I; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D000225:Adenine