Oxygen regulation of nifA transcription in vitro.
Oxygen regulation of nifA transcription in vitro.
复制标题
体外 nifA 转录的氧调节。
DOI：
10.1073/pnas.90.8.3506
复制
发表时间：
1993
影响因子：
11.1
通讯作者：
Helinski,DR
中科院分区：
文献类型：
--
作者：
Agron,PG;Ditta,GS;Helinski,DR
在苜蓿根瘤菌中，固氮关键调控基因nifA和fixK的转录通过FixL和FixJ蛋白的作用而被诱导以响应微好氧。这两种蛋白质是传感器和调节器的同源物，分别是一个大家族的细菌双组分系统参与传感和响应环境变化。膜蛋白FixL的可溶性截短形式FixL* 已被证明是一种血红素蛋白，其响应于氧张力而使FixJ磷酸化和去磷酸化。在这里，我们使用一个体外转录系统来证明，FixJ是一个转录激活因子的nifA和fixK和磷酸化的FixJ显着增加其活性。通过将FixJ与FixL* 和ATP预孵育或通过将FixJ暴露于无机磷酸供体氨基磷酸氢铵来实现磷酸化。FixJ和FixJ-磷酸盐均在所用测定条件下形成肝素抗性复合物。最后，我们能够证明，在FixL* 和ATP存在下，厌氧培养极大地刺激了大肠杆菌或R.苜蓿RNA聚合酶。这种使用大气中的氧来控制nifA转录在体外代表了细菌的双组分信号转导系统的重建在其整体上，从效应到最终的目标，通过使用纯化的组件。
In Rhizobium meliloti, transcription of the key nitrogen-fixation regulatory genes nifA and fixK is induced in response to microaerobiosis through the action of the FixL and FixJ proteins. These two proteins are sensor and regulator homologues, respectively, of a large family of bacterial two-component systems involved in sensing and responding to environmental changes. A soluble, truncated form of the membrane protein FixL, FixL*, has been shown to be a hemoprotein that phosphorylates and dephosphorylates FixJ in response to oxygen tension. Here we use an in vitro transcription system to prove that FixJ is a transcriptional activator of both nifA and fixK and that phosphorylation of FixJ markedly increases its activity. Phosphorylation was achieved either by preincubating FixJ with FixL* and ATP or by exposing FixJ to the inorganic phospho donor ammonium hydrogen phosphoramidate. Both FixJ and FixJ-phosphate formed heparin-resistant complexes under the assay conditions used. Lastly, we were able to show that anaerobiosis, in the presence of FixL* and ATP, greatly stimulates FixJ activity at the nifA promoter with either Escherichia coli or R. meliloti RNA polymerase. This use of atmospheric oxygen to control nifA transcription in vitro represents a reconstitution of a bacterial two-component signal transduction system in its entirety, from effector to ultimate target, by the use of purified components.