González-Pérez M M, Ramos J L, Gallegos M T, Marqués S
Department of Biochemistry, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apartado de Correos 419, E-18008 Granada, Spain.
J Biol Chem. 1999 Jan 22;274(4):2286-90. doi: 10.1074/jbc.274.4.2286.
The Pm promoter, dependent on TOL plasmid XylS regulator, which is activated by benzoate effectors, drives transcription of the meta-cleavage pathway for the metabolism of alkylbenzoates. This promoter is unique in that in vivo transcription is mediated by RNA-polymerase with different sigma factors. In vivo footprinting analysis shows that XylS interacted with nucleotides in the -40 to -70 region. In vivo and in vitro methylation of Pm shows extensive methylation of T at position -42 in the bottom strand, suggesting that it represents a key distortion point that may favor XylS/RNA polymerase interactions. Methylation of T-42 was highest in cells bearing XylS and in the presence of an effector. Gs in the -47 to -61 region appeared to be more protected in cells harboring XylS in the presence than in the absence of the effector. Almost 100 mutants in the Pm region between -41 and -78 were generated; transcriptional analysis of these mutants defined the XylS target as two direct repeats with the sequence TGCAN6GGNCA. These motifs cover the -70 to -56 and the -49 to -35 regions. Single point mutations revealed that nucleotides located at -49 to -46 and at -59, -60, -62, and -70 are the most critical for appropriate XylS-Pm interactions.
Pm启动子依赖于TOL质粒的XylS调节因子，该调节因子可被苯甲酸效应物激活，驱动烷基苯甲酸代谢的间位裂解途径的转录。该启动子的独特之处在于，体内转录由具有不同σ因子的RNA聚合酶介导。体内足迹分析表明，XylS与-40至-70区域的核苷酸相互作用。Pm的体内和体外甲基化显示，底部链中-42位的T发生广泛甲基化，这表明它代表了一个关键的扭曲点，可能有利于XylS/RNA聚合酶的相互作用。在携带XylS的细胞中以及存在效应物的情况下，T-42的甲基化程度最高。在存在效应物的情况下，与不存在效应物相比，-47至-61区域的Gs在携带XylS的细胞中似乎受到更多保护。在-41至-78之间的Pm区域产生了近100个突变体；对这些突变体的转录分析将XylS靶标定义为两个直接重复序列，序列为TGCAN6GGNCA。这些基序覆盖-70至-56区域和-49至-35区域。单点突变表明，位于-49至-46以及-59、-60、-62和-70的核苷酸对于适当的XylS-Pm相互作用最为关键。
