Complex interplay between the LysR-type regulator AtzR and its binding site mediates atzDEF activation in response to two distinct signals.
AtzR is a LysR-type regulator responsible for activation of the cyanuric acid utilization operon atzDEF. AtzR binds the PatzDEF promoter region at a strong recognition element, designated the repressor binding site, and a weaker binding determinant, the activator binding site (ABS). AtzR activates transcription in response to two dissimilar signals, nitrogen limitation and cyanuric acid. In the present work we analyse the structure and function of the cis-acting elements involved in AtzR activation of atzDEF. Hydroxyl radical footprinting assays revealed that the ABS is composed of three functional subsites spaced at one helix-turn intervals. Two modes of interaction with the ABS are detected in vitro: AtzR binds at the ABS-2 and ABS-3 subsites in the absence of inducer, and relocates to interact with the ABS-1 and ABS-2 subsites in the presence of cyanuric acid. In vivo mutational analysis indicates that ABS-1 and ABS-2 are required for full PatzDEF activation in all conditions. In contrast, ABS-3 acts as a 'subunit trap' that hinders productive AtzR interactions with ABS-1 and ABS-2. Our results strongly suggest an activation model in which cyanuric acid and nitrogen limitation cooperate to reposition AtzR from an inactive, ABS-3 bound configuration to an active, ABS-1- and ABS-2-bound configuration.
Porrúa
Odil
O
Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/CSIC. Carretera de Utrera, Km. 1. 41013 Sevilla, Spain.
Platero
Ana Isabel
AI
Santero
Eduardo
E
Del Solar
Gloria
G
Govantes
Fernando
F
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016958:Pseudomonas putida / Q000235:genetics / Q000502:physiology*; D015534:Trans-Activators / Q000378:metabolism*; D014227:Triazines / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D015534:Trans-Activators; D014227:Triazines; D001616:beta-Galactosidase; C004632:cyanuric acid; D009584:Nitrogen