The trigger enzyme PepA (aminopeptidase A) of Escherichia coli, a transcriptional repressor that generates positive supercoiling

Abstract

Escherichia coli aminopeptidase A (PepA) is a trigger enzyme endowed with catalytic activity and DNA-binding properties prominent in transcriptional regulation and site-specific DNA recombination. The current work demonstrates that PepA is a repressor in its own right, capable of specifically inhibiting transcription initiation at promoter P1 of the carAB operon, encoding carbamoylphosphate synthase. Furthermore, in vitro topology studies performed with DNA minicircles demonstrate that PepA binding constrains a single positive supercoil in the carP1 control region. Such a topological event is understood to constitute an impediment to transcription initiation and may serve as a mechanism to regulate gene expression.

Diversity, versatility and complexity of bacterial gene regulation mechanisms: opportunities and drawbacks for applications in synthetic biology

“…DNA looping may be facilitated by a DNA-bending protein such as IHF (Moitoso de Vargas, Kim and Landy 1989). DNA wrapping around hexameric PepA (aminopeptidase A, a trigger enzyme) generates a positive toroidal supercoil and represses transcription initiation at the P1 promoter of the E. coli carAB operon (Nguyen Le Minh, Nadal and Charlier 2016). Repression can also be accomplished by a TF acting as ‘anti-activator’, either by directly competing with the activator for binding to overlapping targets or by blocking the activating signal.…”

“…PepA acts as a strong repressor of P1 activity (Charlier et al. 1995b; Nguyen Le Minh, Nadal and Charlier 2016). It is required for the PurR-mediated purine-specific repression (Devroede et al.…”

“…It is required for the PurR-mediated purine-specific repression (Devroede et al. 2004) but abolishes the stimulatory effect of unliganded RutR (Nguyen Le Minh, Nadal and Charlier 2016). Finally, PepA also appears to be involved in the recruitment of PyrH to the P1 control region by protein–protein contacts (Kholti et al.…”

Regulation of carbamoylphosphate synthesis in Escherichia coli: an amazing metabolite at the crossroad of arginine and pyrimidine biosynthesis

“…This region comprises the binding sites for two transcriptional regulators, RutR and PurR, which equally interact with the P1 operator (see below). DNA topology assays indicated that wrapping of the P1 operator DNA around hexameric PepA generates a positive toroidal supercoil (Nguyen Le Minh et al 2016). In view of the ligand-independent DNA binding of PepA, its multifunctional character, and the structural deformations, PepA was originally supposed to play a merely architectural role in P1 regulation.…”

“…In view of the ligand-independent DNA binding of PepA, its multifunctional character, and the structural deformations, PepA was originally supposed to play a merely architectural role in P1 regulation. However, recently single-round in vitro transcription assays clearly demonstrated that PepA is a repressor in its own right, and hence a trigger enzyme, that specifically inhibits transcription initiation at P1, and the associated topological change is an integral part of the regulatory process (Nguyen Le Minh et al 2016). …”