Transcriptional regulation van het Escherichia coli carAB operon dat codeert voor de carbamoylphosphaatsynthase: de rol van PepA, RutR, Fis en DNA remodeling.

The carAB operon, encoding the unique carbamoyl phosphate synthase of E. coli, occupies a key position in the cellular metabolism, at the crossroad of the arginine and pyrimidine biosynthetic pathways. This particular situation is reflected in the intricate regulatory mechanisms of carAB transcription that is initiated at two promoters in tandem. Regulation of the downstream promoter P2 by arginine-bound ArgR is well known. In contrast, our knowledge of the molecular mechanisms that govern pyrimidine and purine-specific regulation of PI activity is still incomplete. At least six proteins, including allosteric transcriptional regulators (PurR, RutR), small nucleoid-associated proteins (IHF, Fis) and multifunctional trigger enzymes (PepA, PyrH) combined with important DNA remodelling by bending and wrapping appear to be involved in the modulation of PI activity. In this project we will: (i) unravel the precise topology of regulatory PepA-DNA complexes, (ii) analyse PepAPyrH protein-protein contacts, (iii) determine the contribution and the mode of action of the uracil-sensitive allosteric transcriptional regulator RutR in the control of PI activity, and (iv) dissect the interaction of the nucleoid associated factor Fis (factor for inversion ~timulation) with the carAB control region and analyze the concomitant effects on PI and P2 activity in vitro and in vivo.