An unusual repressor controls the expression of a crucial nicotine-degrading gene cluster in Pseudomonas putida S16
An unusual repressor controls the expression of a crucial nicotine-degrading gene cluster in Pseudomonas putida S16
复制标题
一种不寻常的阻遏蛋白控制着恶臭假单胞菌 S16 中关键的尼古丁降解基因簇的表达
DOI：
10.1111/mmi.12533
复制
发表时间：
2014
影响因子：
3.6
通讯作者：
Xu Ping
中科院分区：
文献类型：
--
作者：
Wang Lijuan;Tang Hongzhi;Yu Hao;Yao Yuxiang;Xu Ping
含有螺旋-转角-螺旋（HTH）DNA结合结构域的转录因子广泛存在于细菌中，用于按需调节基因表达，并作为结合回文DNA片段的同源二聚体发挥作用。在这里，我们表明，含HTH的转录调节因子NicR 2在恶臭假单胞菌S16中控制尼古丁降解中关键基因簇（Nic 2）的表达中起着关键作用，NicR 2以不同于大多数其他使用HTH识别的DNA结合蛋白的方式结合DNA。电泳迁移率变动分析（EMSA）和DNA酶I足迹分析表明，NicR 2与thenic 2启动子区的一个28 bp的反向重复序列（IR）直接相互作用。使用合成DNA片段的EMSA，我们发现NicR 2二聚体和四聚体都可以结合到IR的半位点上。这一点通过生物层干涉测量和交联实验得到了独立的证实。我们的研究结果表明，两个NicR 2二聚体通过蛋白质-蛋白质相互作用协同结合IR，每个二聚体结合IR的半位点。因此，NicR 2似乎是一种不寻常的调节剂，它使用HTH进行识别，并显示出一些使用β折叠的调节剂的结合特征。假单胞菌中尼古丁降解的转录调控突出了原核转录调控的复杂性。
Transcriptional factors that contain helix–turn–helix (HTH) DNA‐binding domains are widespread in bacteria for regulating gene expression on demand, and function as homodimers that bind a palindromic DNA segment. Here, we show that an HTH‐containing transcriptional regulator, NicR2, inPseudomonas putidaS16 plays a critical role in controlling the expression of a crucial gene cluster (nic2) in nicotine degradation, and NicR2 binds DNA in a manner different from most other DNA‐binding proteins that use HTHs for recognition. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting indicate that NicR2 directly interacts with a 28 bp inverted repeat (IR) in thenic2 promoter region. Using EMSA with synthetic DNA fragments, we found that both NicR2 dimer and tetramer can bind to the half‐site of the IR. This is confirmed independently by biolayer interferometry and cross‐linking experiments. Our results indicate that two NicR2 dimers bind to the IR cooperatively through protein–protein interactions, with each dimer binding the half‐site of the IR. Thus, NicR2 appears to be an unusual regulator, which uses HTH for recognition and displays the binding characteristics of some regulators that useβ‐sheets. The transcriptional regulation of nicotine degradation inPseudomonashighlights a new level of complexity in prokaryotic transcriptional regulation.