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Activation and repression of a σ N ‐dependent promoter naturally lacking upstream activation sequences
Author(s) -
Porrúa Odil,
GarcíaGonzález Vicente,
Santero Eduardo,
Shingler Victoria,
Govantes Fernando
Publication year - 2009
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2009.06779.x
Subject(s) - psychological repression , biology , transcription (linguistics) , operon , repressor , promoter , upstream activating sequence , microbiology and biotechnology , transcription factor , dna footprinting , in vitro , dna binding protein , genetics , gene , gene expression , mutant , linguistics , philosophy
Summary 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 σ N ‐dependent promoter P atzR , activated by NtrC and repressed by AtzR. Here we use in vivo and in vitro approaches to address the mechanisms of P atzR activation and repression. Activation by NtrC did not require any promoter sequences other than the σ N recognition motif both in vivo and in vitro , suggesting that NtrC activates P atzR 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 P atzR promoter in vitro correlates with AtzR affinity for its binding site. In addition, AtzR prevents transcription from P atzR when added to a preformed E‐σ N –P atzR 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‐σ N are mutually exclusive. Taken together, these results strongly support the notion that AtzR represses transcription from P atzR by competing with E‐σ N for their overlapping binding sites. There are no previous reports of a similar mechanism for repression of σ N ‐dependent transcription.