An <scp>A</scp>‐tract at the <scp>AtzR</scp> binding site assists <scp>DNA</scp> binding, inducer‐dependent repositioning and transcriptional activation of the <scp>P<i>atzDEF</i></scp> promoter

<jats:title>Summary</jats:title><jats:p>The <jats:styled-content style="fixed-case">LysR</jats:styled-content>‐type regulator <jats:styled-content style="fixed-case">AtzR</jats:styled-content> activates the <jats:italic>Pseudomonas</jats:italic> sp. <jats:styled-content style="fixed-case">ADP <jats:italic>atzDEF</jats:italic></jats:styled-content> operon in response to nitrogen limitation and cyanuric acid. Activation involves repositioning of the <jats:styled-content style="fixed-case">AtzR</jats:styled-content> tetramer on the <jats:styled-content style="fixed-case">P<jats:italic>atzDEF</jats:italic></jats:styled-content> promoter and relaxation of an <jats:styled-content style="fixed-case">AtzR</jats:styled-content>‐induced <jats:styled-content style="fixed-case">DNA</jats:styled-content> bend. Here we examine the <jats:italic>in vivo</jats:italic> and <jats:italic>in vitro</jats:italic> contribution of an <jats:styled-content style="fixed-case">A</jats:styled-content><jats:sub>5</jats:sub>‐tract present at the <jats:styled-content style="fixed-case">P<jats:italic>atzDEF</jats:italic></jats:styled-content> promoter region to <jats:styled-content style="fixed-case">AtzR</jats:styled-content> binding and transcriptional activation. Substitution of the <jats:styled-content style="fixed-case">A</jats:styled-content>‐tract for the sequence <jats:styled-content style="fixed-case">ACTCA</jats:styled-content> prevented <jats:styled-content style="fixed-case">P<jats:italic>atzDEF</jats:italic></jats:styled-content> activation and high‐affinity <jats:styled-content style="fixed-case">AtzR</jats:styled-content> binding, impaired <jats:styled-content style="fixed-case">AtzR</jats:styled-content> contacts with the activator binding site and shifted the position of the <jats:styled-content style="fixed-case">AtzR</jats:styled-content>‐induced <jats:styled-content style="fixed-case">DNA</jats:styled-content> bend. Analysis of a collection of mutants bearing different alterations in the <jats:styled-content style="fixed-case">A</jats:styled-content>‐tract sequence showed that the extent of <jats:styled-content style="fixed-case">AtzR</jats:styled-content>‐dependent activation does not correlate with the magnitude or orientation of the spontaneous <jats:styled-content style="fixed-case">DNA</jats:styled-content> bend generated at this site. Our results support the notion that indirect readout of the <jats:styled-content style="fixed-case">A</jats:styled-content>‐tract‐associated narrow minor groove is essential for the <jats:styled-content style="fixed-case">AtzR</jats:styled-content>–<jats:styled-content style="fixed-case">DNA</jats:styled-content> complex to achieve a conformation competent for activation of the <jats:styled-content style="fixed-case">P<jats:italic>atzDEF</jats:italic></jats:styled-content> promoter. Conservation of this motif in several binding sites of <jats:styled-content style="fixed-case">LysR</jats:styled-content>‐type regulators suggests that this mechanism may be shared by other proteins in this family.</jats:p>