Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/Consejo Superior de Investigaciones Científicas/Junta de Andalucía, Carretera de Utrera, Km. 1, 41013, Sevilla, Spain; Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, Carretera de Utrera, Km. 1, 41013, Sevilla, Spain.
Mol Microbiol. 2013 Oct;90(1):72-87. doi: 10.1111/mmi.12346. Epub 2013 Aug 15.
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 PatzDEF promoter and relaxation of an AtzR-induced DNA bend. Here we examine the in vivo and in vitro contribution of an A5 -tract present at the PatzDEF promoter region to AtzR binding and transcriptional activation. Substitution of the A-tract for the sequence ACTCA prevented PatzDEF 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 PatzDEF 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.
LysR 型调控蛋白 AtzR 响应氮限制和三聚氰胺激活假单胞菌 ADP atzDEF 操纵子。激活涉及 AtzR 四聚体在 PatzDEF 启动子上的重新定位和 AtzR 诱导的 DNA 弯曲的松弛。在这里，我们研究了 PatzDEF 启动子区域存在的 A 链段在 AtzR 结合和转录激活中的体内和体外贡献。用序列 ACTCA 替代 A 链段可防止 PatzDEF 激活和高亲和力 AtzR 结合，损害 AtzR 与激活剂结合位点的接触，并改变 AtzR 诱导的 DNA 弯曲的位置。对一系列具有不同 A 链段序列改变的突变体的分析表明，AtzR 依赖性激活的程度与该位点产生的自发 DNA 弯曲的幅度或方向无关。我们的结果支持这样一种观点，即间接读取与 A 链段相关的狭窄小沟是 AtzR-DNA 复合物实现激活 PatzDEF 启动子的构象所必需的。这种基序在几个 LysR 型调控蛋白的结合位点中保守，表明该机制可能被该家族中的其他蛋白共享。
