Phosphorylation, dephosphorylation and DNA-binding of the Bradyrhizobium japonicum RegSR two-component regulatory proteins.
Phosphorylation, dephosphorylation and DNA-binding of the Bradyrhizobium japonicum RegSR two-component regulatory proteins.
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日本慢生根瘤菌 RegSR 双组分调节蛋白的磷酸化、去磷酸化和 DNA 结合。
DOI：
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
H. Fischer
中科院分区：
文献类型：
--
作者：
R. Emmerich;K. Panglungtshang;P. Strehler;H. Hennecke;H. Fischer
在低氧条件下，慢生根瘤菌固氮所需的许多基因的诱导依赖于FixR-NifA操纵子中编码的氧化还原反应转录激活因子NIFA。该操纵子的基础表达依赖于反应调节因子RegR和位于FixR-NIFA启动子区域-68位附近的DNA元件。为了研究RegR的功能特性及其与其推定的同源蛋白激酶regs的相互作用，我们过量生产并亲和纯化了regR和regs的一个截短的可溶变异体(regs(C))，它们都是N端His(6)标记的蛋白质。当与[Gamma-(32)P]ATP孵育时，RegS(C)会自动磷酸化，并催化将磷酸化标记转移到RegR。磷酸化形式的RegS(C)对RegR-磷酸具有磷酸酶活性。化学稳定性试验和定点突变分别鉴定了RegS和RegR的H_219和D_(63)氨基酸为磷酸化残基。与孤立结构域的竞争实验表明，RegR的N末端结构域而不是C末端结构域与Regs(C)相互作用。带移实验表明，与去磷酸化的RegR或不能磷酸化的突变蛋白RegR-D63N相比，磷酸化的RegR具有至少8倍的DNA结合活性。总之，日本血吸虫的RegSR蛋白在体外表现出典型的双组分调控系统的功能特性。
Under low oxygen conditions, induction of many genes required for nitrogen fixation in Bradyrhizobium japonicum depends on the redox-responsive transcriptional activator NifA which is encoded in the fixR-nifA operon. Basal expression of this operon depends on the response regulator RegR and a DNA element located around position -68 in the fixR-nifA promoter region. To investigate the functional properties of RegR and the interaction with its putative cognate kinase, RegS, we overproduced and affinity-purified RegR and a truncated soluble variant of RegS (RegS(C)), both as N-terminally His(6)-tagged proteins. RegS(C) autophosphorylated when incubated with [gamma-(32)P]ATP, and it catalyzed the transfer of the phosphoryl label to RegR. The phosphorylated form of RegS(C) exhibited phosphatase activity on RegR-phosphate. Chemical stability tests and site-specific mutagenesis identified amino acids H219 and D63 of RegS and RegR, respectively, as the phosphorylated residues. Competition experiments with isolated domains demonstrated that the N-terminal but not the C-terminal domain of RegR interacts with RegS(C). Band-shift experiments revealed that phosphorylated RegR had at least eightfold enhanced DNA-binding activity compared with dephosphorylated RegR or the mutant protein RegR-D63N, which cannot be phosphorylated. In conclusion, the RegSR proteins of B. japonicum exhibit functional properties in vitro that are typical of two-component regulatory systems.
DOI：
10.1073/pnas.85.14.4976
发表时间：
1988-07-01
影响因子：
11.1
作者：
KEENER, J;KUSTU, S
通讯作者：
KUSTU, S
硝酸盐传感系统的纯化成分 NarX 和 NarL 在体外催化磷酸化和去磷酸化。
DOI：
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发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Walker,MS;DeMoss,JA
通讯作者：
DeMoss,JA