title: Purine and pyrimidine-specific repression of the Escherichia coli carAB operon are functionally and structurally coupled.
abstract: Transcription of the carAB operon encoding the sole carbamoylphosphate synthetase of Escherichia coli proceeds from a tandem pair of promoters. P2, downstream, is repressed by arginine and the ArgR protein, whereas P1 is submitted to pyrimidine-specific regulation and as shown here to purine-specific control exerted by binding of the PurR protein to a PUR box sequence centered around nucleotide -128.5 with respect to the start of P1 transcription. In vivo analyses of the effects of trans and cis-acting mutations on the regulatory responses and single round in vitro transcription assays indicated that ligand-bound PurR is by itself unable to inhibit P1 promoter activity. To exert its effect PurR relies on the elaborated nucleoprotein complex that governs P1 activity in a pyrimidine-specific manner. Thus we reveal the existence of an unprecedented functional and structural coupling between the modulation of P1 activity by purine and pyrimidine residues that appears to result from the unique position of the PUR box in the carAB control region, far upstream of the promoter. Missing contact and premethylation binding interference studies revealed the importance of base-specific groups and of structural aspects of the PUR box sequence in complex formation. Permutation assays indicated that the overall PurR-induced bending of the carAB control region is slightly less pronounced than that of the purF operator. The PUR boxes of the carAB operon of E.coli and Salmonella typhimurium are unique in that they have a guanine residue at position eight. Interestingly, guanine at this position has been proposed to be extremely unfavorable on the basis of modeling and binding studies, as its exocyclic amino group would enter into a steric clash with the side-chain of lysine 55. To analyze the effect of guanine at position eight in the upstream half-site of the carAB operator we constructed the adenine derivative and assayed in vivo repressibility of P1 promoter activity and in vitroPurR binding to the mutant operator, and constructed a molecular model for the unusual lysine 55-guanine 8 interaction.
authors.affiliation: Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Neel
authors.initials: N
authors.lastname: Devroede
authors.forename: Thia-Lin
authors.initials: TL
authors.lastname: Thia-Toong
authors.forename: Daniel
authors.initials: D
authors.lastname: Gigot
authors.forename: Dominique
authors.initials: D
authors.lastname: Maes
authors.forename: Daniel
authors.initials: D
authors.lastname: Charlier
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011687:Purines / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C065825:PurR protein, Bacteria; C489317:PurR protein, E coli; D011687:Purines; D011743:Pyrimidines; D012097:Repressor Proteins; D004247:DNA; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); C030986:pyrimidine; C030985:purine