Helicobacter pylori NikR's Interaction with DNA: A Two-Tiered Mode of Recognition
Helicobacter pylori NikR's Interaction with DNA: A Two-Tiered Mode of Recognition
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DOI：
10.1021/bi801481j
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发表时间：
2009-01-27
期刊：
影响因子：
2.9
通讯作者：
Michel, Sarah L. J.
中科院分区：
文献类型：
--
作者：
Dosanjh, Nuvjeevan S.;West, Abby L.;Michel, Sarah L. J.
HPNikR 是在有毒细菌幽门螺杆菌中发现的原核镍结合转录因子。 HPNikR 作为激活剂或阻抑剂调节多个基因的表达，包括那些涉及镍离子稳态、酸适应和铁吸收的基因。 HPNikR 调节的基因的目标操纵子序列不包含可识别的对称识别位点，并且 HPNikR 区分其调节的基因的机制尚不清楚。使用竞争性荧光各向异性 (FA) 和电泳凝胶迁移率变动 (EMSA) 测定，对 HPNikR 和直接调控基因的目标操纵基因序列（ureA、NixA、NikR、Fur OPI、Fur OPII、Frpb4、FecA3 和 exbB）之间的相互作用进行了表征。这些研究表明，HPNikR 利用两层 DNA 识别模式，与一些具有高亲和力的基因和其他具有低亲和力的基因结合。受 HPNikR 严格调控的基因编码利用镍的蛋白质，而那些不受严格调控的基因编码其他类型的蛋白质。低亲和力金属离子对第二金属结合位点的亲和力被确定在微摩尔范围内，并且静电对HPNikR-DNA结合事件的贡献被确定。对 HPNikR 和 ureA 之间相互作用的序列长度和同一性的作用的详细研究揭示了 DNA 结合的特定长度要求。
HPNikR is a prokaryotic nickel binding transcription factor found in the virulent bacterium Helicobacter pylori. HPNikR regulates the expression of multiple genes as an activator or repressor, including those involved in nickel ion homeostasis, acid adaptation, and iron uptake. The target operator sequences of the genes regulated by HPNikR do not contain identifiable symmetrical recognition sites, and the mechanism by which HPNikR distinguishes between the genes it regulates is not understood. Using competitive fluorescence anisotropy (FA) and electrophoretic gel mobility shift (EMSA) assays, the interactions between HPNikR and the target operator sequences of the genes directly regulated (ureA, NixA, NikR, Fur OPI, Fur OPII, Frpb4, FecA3, and exbB) were characterized. These studies revealed that HPNikR utilizes a two-tiered mode of DNA recognition by binding to some genes with high affinity and others with low affinity. The genes that are tightly regulated by HPNikR encode proteins that utilize nickel, while those that are less tightly regulated encode other types of proteins. The affinities of low-affinity metal ions for a second metal binding site were determined to be in the micromolar regime, and a contribution of electrostatics to the HPNikR-DNA binding event was determined. Detailed studies of the role of sequence length and identity for the interaction between HPNikR and ureA revealed a specific length requirement for DNA binding.