Ruiz R, Ramos J L, Egan S M
Consejo Superior de Investigaciones Cientificas, Estación Experimental del Zaidín, Department of Plant Biochemistry, Albareda, Granada, Spain.
FEBS Lett. 2001 Mar 2;491(3):207-11. doi: 10.1016/s0014-5793(01)02192-5.
The Pseudomonas putida meta-cleavage operon encodes the enzymes for the catabolism of alkylbenzoates. Activation of meta-operon transcription is mediated by the XylS protein which, upon activation by effectors, binds two sites between -70 and -35 with respect to the main transcription initiation point at the Pm promoter. Two naturally occurring regulators, XylS and XylS1, that differ by only five amino acids, have been analyzed with regard to potential interactions of these positive regulators with the C-terminal domain of the alpha subunit of RNA polymerase (alpha-CTD). For these studies we expressed a derivative of alpha deprived of the entire C-terminal domain (alpha-Delta235) and found that expression from Pm with XylS or XylS1 was significantly decreased. To discern whether alpha-CTD activation depended on interactions with DNA and/or XylS proteins we tested a large collection of alanine substitutions within alpha-CTD. Most substitutions that had an effect on XylS and XylS1-dependent transcription were located in or adjacent to helix 1 and 4, which are known to be involved in alpha-CTD interactions with DNA. Two alanine substitutions in helix 3 (residues 287 and 291) identified a putative region of alpha-CTD/XylS regulator interactions.
恶臭假单胞菌间位裂解操纵子编码用于烷基苯甲酸分解代谢的酶。间位操纵子转录的激活由XylS蛋白介导，该蛋白在效应物激活后，在相对于Pm启动子处的主要转录起始点的-70至-35之间结合两个位点。两种仅相差五个氨基酸的天然存在的调节因子XylS和XylS1，已就这些正调节因子与RNA聚合酶α亚基的C末端结构域（α-CTD）的潜在相互作用进行了分析。对于这些研究，我们表达了缺失整个C末端结构域的α衍生物（α-Delta235），并发现使用XylS或XylS1时Pm的表达显著降低。为了辨别α-CTD激活是否依赖于与DNA和/或XylS蛋白的相互作用，我们测试了α-CTD内大量的丙氨酸取代。大多数对XylS和XylS1依赖性转录有影响的取代位于螺旋1和4内或附近，已知它们参与α-CTD与DNA的相互作用。螺旋3中的两个丙氨酸取代（残基287和291）确定了α-CTD/XylS调节因子相互作用的一个假定区域。
