Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators
Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators
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DOI：
10.1074/jbc.m310041200
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发表时间：
2004-02-13
影响因子：
4.8
通讯作者：
Whitelaw, ML
中科院分区：
文献类型：
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作者：
Chapman-Smith, A;Lutwyche, JK;Whitelaw, ML
碱性螺旋-环-螺旋（bHLH） PAS转录调控因子控制关键的发育和代谢过程，包括对缺氧和环境污染物等刺激的转录反应，分别由缺氧诱导因子（hif - α）和二恶英（芳烃）受体（DR）介导。bHLH蛋白含有一个与螺旋-环-螺旋二聚化结构域相邻的基本DNA结合序列。bHLH的二聚化。PAS蛋白还受PAS区域的调控，PAS区域控制着伴侣选择的特异性，因此hif - α和DR必须与芳烃核转运子（Arnt）二聚才能形成功能性的DNA结合复合物。在这里，我们分析了含有n端bHLH和bHLH的纯化细菌表达蛋白。Arnt， DR和HIF-1alpha的PAS区域，并评估PAS结构域对体外DNA结合的贡献。bHLH的共表达显著增强了细菌中功能性DNA结合蛋白的恢复。DR或HIF-1alpha的PAS区域与Arnt的相应区域。DR/Arnt和HIF-1alpha/Arnt异源二聚体与其同源DNA序列形成稳定的蛋白-DNA复合物需要PAS A结构域，K-D值分别为0.4 nM和接近50 nM。相比之下，Arnt的PAS结构域的存在对Arnt同型二聚体结合DNA的影响很小，这些DNA结合的K-D为45 nM。在DR的情况下，高亲和力的DNA结合和二聚体的稳定性都是特异性的原生PAS结构域，因为嵌合体中PAS a结构域被Arnt的等效结构域取代，产生了不稳定的蛋白质，结合DNA很差。
The basic helix-loop-helix (bHLH) PAS transcriptional regulators control critical developmental and metabolic processes, including transcriptional responses to stimuli such as hypoxia and environmental pollutants, mediated respectively by hypoxia inducible factors (HIF-alpha) and the dioxin (aryl hydrocarbon) receptor (DR). The bHLH proteins contain a basic DNA binding sequence adjacent to a helix-loop-helix dimerization domain. Dimerization among bHLH. PAS proteins is additionally regulated by the PAS region, which controls the specificity of partner choice such that HIF-alpha and DR must dimerize with the aryl hydrocarbon nuclear translocator (Arnt) to form functional DNA binding complexes. Here, we have analyzed purified bacterially expressed proteins encompassing the N-terminal bHLH and bHLH. PAS regions of Arnt, DR, and HIF-1alpha and evaluated the contribution of the PAS domains to DNA binding in vitro. Recovery of functional DNA binding proteins from bacteria was dramatically enhanced by coexpression of the bHLH. PAS regions of DR or HIF-1alpha with the corresponding region of Arnt. Formation of stable protein-DNA complexes by DR/Arnt and HIF-1alpha/Arnt heterodimers with their cognate DNA sequences required the PAS A domains and exhibited K-D values of 0.4 nM and similar to50 nM, respectively. In contrast, the presence of the PAS domains of Arnt had little effect on DNA binding by Arnt homodimers, and these bound DNA with a K-D of 45 nM. In the case of the DR, both high affinity DNA binding and dimer stability were specific to its native PAS domain, since a chimera in which the PAS A domain was substituted with the equivalent domain of Arnt generated a destabilized protein that bound DNA poorly.