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An A ‐tract at the AtzR binding site assists DNA binding, inducer‐dependent repositioning and transcriptional activation of the P atzDEF promoter
Author(s) -
Porrúa Odil,
LópezSánchez Aroa,
Platero Ana I.,
Santero Eduardo,
Shingler Victoria,
Govantes Fernando
Publication year - 2013
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/mmi.12346
Subject(s) - biology , binding site , operon , dna , consensus sequence , dna binding protein , promoter , activator (genetics) , repressor , microbiology and biotechnology , biochemistry , transcription factor , mutant , gene , gene expression , base sequence
Summary The LysR ‐type regulator AtzR activates the Pseudomonas sp. ADP atzDEF operon in response to nitrogen limitation and cyanuric acid. Activation involves repositioning of the AtzR tetramer on the P atzDEF promoter and relaxation of an AtzR ‐induced DNA bend. Here we examine the in vivo and in vitro contribution of an A 5 ‐tract present at the P atzDEF promoter region to AtzR binding and transcriptional activation. Substitution of the A ‐tract for the sequence ACTCA prevented P atzDEF activation and high‐affinity AtzR binding, impaired AtzR contacts with the activator binding site and shifted the position of the AtzR ‐induced DNA bend. Analysis of a collection of mutants bearing different alterations in the A ‐tract sequence showed that the extent of AtzR ‐dependent activation does not correlate with the magnitude or orientation of the spontaneous DNA bend generated at this site. Our results support the notion that indirect readout of the A ‐tract‐associated narrow minor groove is essential for the AtzR – DNA complex to achieve a conformation competent for activation of the P atzDEF promoter. Conservation of this motif in several binding sites of LysR ‐type regulators suggests that this mechanism may be shared by other proteins in this family.