Dosanjh Nuvjeevan S, West Abby L, Michel Sarah L J
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201-1180, USA.
Biochemistry. 2009 Jan 27;48(3):527-36. doi: 10.1021/bi801481j.
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.
HPNikR是一种在致病性细菌幽门螺杆菌中发现的原核镍结合转录因子。HPNikR作为激活剂或阻遏物调节多个基因的表达，包括那些参与镍离子稳态、酸适应和铁摄取的基因。HPNikR调控的基因的靶操纵序列不包含可识别的对称识别位点，并且HPNikR区分其调控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）分析，对HPNikR与直接调控基因（ureA、NixA、NikR、Fur OPI、Fur OPII、Frpb4、FecA3和exbB）的靶操纵序列之间的相互作用进行了表征。这些研究表明，HPNikR通过以高亲和力结合一些基因和以低亲和力结合其他基因来利用两级DNA识别模式。受HPNikR严格调控的基因编码利用镍的蛋白质，而那些调控不那么严格的基因编码其他类型的蛋白质。确定了低亲和力金属离子对第二个金属结合位点的亲和力处于微摩尔范围，并确定了静电对HPNikR-DNA结合事件的贡献。对HPNikR与ureA相互作用中序列长度和同一性作用的详细研究揭示了DNA结合的特定长度要求。
