Solution structure of the C-terminal transcriptional activator domain of FixJ from Sinorhizobium meliloti and its recognition of the fixK promoter
Solution structure of the C-terminal transcriptional activator domain of FixJ from Sinorhizobium meliloti and its recognition of the fixK promoter
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DOI：
10.1021/bi0509043
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发表时间：
2005-11-15
期刊：
影响因子：
2.9
通讯作者：
Shiro, Y
中科院分区：
文献类型：
--
作者：
Kurashima-Ito, K;Kasai, Y;Shiro, Y
FixJ是一种应答调节蛋白，参与苜蓿中华根瘤菌固氮基因转录激活的双组分信号转导途径。磷酸化后，FixJ可转录激活fixK和nifA启动子。我们通过凝胶迁移率变动分析，在fixK启动子的高亲和力结合位点鉴定出一个16碱基对的FixJ识别序列。此外，利用核磁共振波谱法解析了FixJ截短的C端DNA结合结构域（FixJC）的溶液结构。FixJC包含5个α -螺旋，编码一个典型的螺旋 - 转角 - 螺旋基序，作为潜在的DNA结合核心，与NarL的C端DNA结合结构域具有最高的结构相似性。在FixJC的核磁共振谱中，加入含有高亲和力FixJ结合位点识别序列的DNA片段，会在核磁共振时间尺度上产生中等至缓慢的交换相互作用，而对照DNA的交换则很快。这些光谱数据表明，尽管FixJC在溶液中以单体形式存在，但不止一个FixJC分子与识别序列结合。这一结果与转录活性形式的FixJ是磷酸化形式的同二聚体这一情形相符。
FixJ is a response regulator of the two-component signal transduction pathway involved in the transcriptional activation of nitrogen fixation genes of Sinorhizobium meliloti. Upon phosphorylation, FixJ transcriptionally activates the fixK and nifA promoters. We identified a FixJ recognition sequence of 16 bp in the high affinity binding site of the fixK promoter by means of a gel shift assay. In addition, the solution structure of the truncated C-terminal DNA binding domain of FixJ (FixJC) was solved by NMR spectroscopy. FixJC contains five a.-helices that encode a typical helix-turn-helix motif as a potential DNA binding core with the highest structural similarity toward the C-terminal DNA binding domain of NarL. The addition of the DNA fragment containing the recognition sequence of the high affinity FixJ binding site resulted in intermediate to slow exchange interactions on the NMR time scale in the spectrum of FixJC, while the exchange was rapid in the case of control DNA. These spectral data suggest that more than one molecule of FixJC binds to the recognition sequence, although FixJC alone is present in monomeric form in solution. This result is consistent with a scenario in which a transcriptionally active species of FixJ is a homodimer of the phosphorylated form.