Activation and repression of a σN-dependent promoter naturally lacking upstream activation sequences

Abstract

The Pseudomonas sp. strain ADP protein AtzR is a LysR-type transcriptional regulator required for activation of the atzDEF operon in response to nitrogen limitation and cyanuric acid. Transcription of atzR is directed by the σN-dependent promoter PatzR, activated by NtrC and repressed by AtzR. Here we use in vivo and in vitro approaches to address the mechanisms of PatzR activation and repression. Activation by NtrC did not require any promoter sequences other than the σN recognition motif both in vivo and in vitro, suggesting that NtrC activates PatzR in an upstream activation sequences-independent fashion. Regarding AtzR-dependent autorepression, our in vitro transcription experiments show that the concentration of AtzR required for repression of the PatzR promoter in vitro correlates with AtzR affinity for its binding site. In addition, AtzR prevents transcription from PatzR when added to a preformed E-σN–PatzR closed complex, but isomerization to an open complex prevents repression. Gel mobility shift and DNase I footprint assays indicate that DNA-bound AtzR and E-σN are mutually exclusive. Taken together, these results strongly support the notion that AtzR represses transcription from PatzR by competing with E-σN for their overlapping binding sites. There are no previous reports of a similar mechanism for repression of σN-dependent transcription.