Bertoni G, Marqués S, de Lorenzo V
Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid, Spain.
Mol Microbiol. 1998 Feb;27(3):651-9. doi: 10.1046/j.1365-2958.1998.00715.x.
The mechanism by which XylR, the toluene-responsive activator of the sigma54-dependent Pu and Ps promoters of the Pseudomonas TOL plasmid pWW0, downregulates its own sigma70 promoter Prhas been examined. An in vitro transcription system was developed in order to reproduce the repression of Probserved in cells of P. putida (pWW0) both in the presence and in the absence of the XylR inducer, benzyl alcohol. DNA templates bearing the two sigma70-RNA polymerase (RNAP) binding sites of Pr, which overlap the upstream activating sequences (UAS) for XylR in the divergent sigma54 promoter Ps, were transcribed in the presence of a constitutively active XylR variant deleted of its N-terminal domain (XylRdeltaA). The addition of ATP, known to trigger multimerization of the regulator at the UAS, enhanced the repression of Pr by XylR. Furthermore, we observed activation of the divergent sigma54 promoter Ps during Pr downregulation by XylRdeltaA. These results support the notion that activation of XylR by aromatic inducers in vivo triggers a transcriptional switch between Pr and Ps. Such a switch is apparently caused by the ATP-dependent multimerization and strong DNA binding of the protein required for activation of the sigma54 promoter. This device could reset the level of XylR expression during activation of the sigma54 Pu and Ps promoters of the TOL plasmid.
已对木糖响应激活因子XylR下调其自身σ⁷⁰启动子Pr的机制进行了研究。XylR是假单胞菌TOL质粒pWW0上依赖σ⁵⁴的Pu和Ps启动子的甲苯响应激活因子。为了重现恶臭假单胞菌（pWW0）细胞在有和没有XylR诱导剂苄醇的情况下所观察到的Pr抑制作用，开发了一种体外转录系统。携带Pr的两个σ⁷⁰-RNA聚合酶（RNAP）结合位点的DNA模板，其与发散型σ⁵⁴启动子Ps中XylR的上游激活序列（UAS）重叠，在存在缺失其N端结构域的组成型活性XylR变体（XylRΔA）的情况下进行转录。已知ATP会触发调节因子在UAS处的多聚化，添加ATP增强了XylR对Pr的抑制作用。此外，我们观察到在XylRΔA下调Pr期间发散型σ⁵⁴启动子Ps的激活。这些结果支持了这样一种观点，即体内芳香族诱导剂对XylR的激活会触发Pr和Ps之间的转录开关。这种开关显然是由σ⁵⁴启动子激活所需蛋白质的ATP依赖性多聚化和强DNA结合引起的。该机制可以在TOL质粒的σ⁵⁴ Pu和Ps启动子激活期间重置XylR表达水平。
