Biochemical Characterization of the Transcriptional Regulator BzdR from Azoarcus sp. CIB
Biochemical Characterization of the Transcriptional Regulator BzdR from Azoarcus sp. CIB
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DOI：
10.1074/jbc.m110.143503
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发表时间：
2010-11-12
影响因子：
4.8
通讯作者：
Carmona, Manuel
中科院分区：
文献类型：
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作者：
Durante-Rodriguez, Gonzalo;Andres Valderrama, J.;Carmona, Manuel
BzdR转录调节子控制着P-N启动子，该启动子负责Azoarcus sp. CIB中苯甲酸盐的厌氧催化，构成了一个新的转录调节子亚家族的原型。在这里，我们提供了一些关于BzdR蛋白的功能结构关系的见解。分析超离心研究表明，BzdR在溶液中是同源二聚体。已获得的BzdR二聚体的电子显微镜三维重建，和每个BzdR单体的相应的N-和C-末端结构域的预测结构可以被安装到这样的重建。凝胶阻滞和超离心实验表明BzdR与其同源启动子的结合是协同的。不同的生化方法表明，效应分子苯甲酰辅酶A诱导BzdR的构象变化，而不影响其寡聚状态。已经通过体外转录测定建立了P-N启动子的BzdR依赖性抑制及其在苯甲酰-CoA存在下的活化。单体BzdR 4和BzdR 5突变体调节剂揭示了BzdR的二聚化对于DNA结合是必需的。值得注意的是，缺乏连接BzdR的N-和C-末端结构域的接头区的BzdR Delta L蛋白也是二聚体的，并且表现为P-N启动子的超级阻遏物。这些数据表明，BzdR的接头区是不是必不可少的蛋白质二聚体，而是需要转移的构象变化诱导的苯甲酰辅酶A的DNA结合结构域，导致的阻遏物的释放。提出了BzdR调节器的作用模型。
The BzdR transcriptional regulator that controls the P-N promoter responsible for the anaerobic catabolism of benzoate in Azoarcus sp. CIB constitutes the prototype of a new subfamily of transcriptional regulators. Here, we provide some insights about the functional-structural relationships of the BzdR protein. Analytical ultracentrifugation studies revealed that BzdR is homodimeric in solution. An electron microscopy three-dimensional reconstruction of the BzdR dimer has been obtained, and the predicted structures of the respective N- and C-terminal domains of each BzdR monomer could be fitted into such a reconstruction. Gel retardation and ultracentrifugation experiments have shown that the binding of BzdR to its cognate promoter is cooperative. Different biochemical approaches revealed that the effector molecule benzoyl-CoA induces conformational changes in BzdR without affecting its oligomeric state. The BzdR-dependent inhibition of the P-N promoter and its activation in the presence of benzoyl-CoA have been established by in vitro transcription assays. The monomeric BzdR4 and BzdR5 mutant regulators revealed that dimerization of BzdR is essential for DNA binding. Remarkably, a BzdR Delta L protein lacking the linker region connecting the N- and C-terminal domains of BzdR is also dimeric and behaves as a super-repressor of the P-N promoter. These data suggest that the linker region of BzdR is not essential for protein dimerization, but rather it is required to transfer the conformational changes induced by the benzoyl-CoA to the DNA binding domain leading to the release of the repressor. A model of action of the BzdR regulator has been proposed.