Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
Nucleic Acids Res. 2020 Mar 18;48(5):2199-2208. doi: 10.1093/nar/gkaa056.
Microorganisms use zinc-sensing regulators to alter gene expression in response to changes in the availability of zinc, an essential micronutrient. Under zinc-replete conditions, the Fur-family metalloregulator Zur binds to DNA tightly in its metallated repressor form to Zur box operator sites, repressing the transcription of zinc uptake transporters. Derepression comes from unbinding of the regulator, which, under zinc-starvation conditions, exists in its metal-deficient non-repressor forms having no significant affinity with Zur box. While the mechanism of transcription repression by Zur is well-studied, little is known on how derepression by Zur could be facilitated. Using single-molecule/single-cell measurements, we find that in live Escherichia coli cells, Zur's unbinding rate from DNA is sensitive to Zur protein concentration in a first-of-its-kind biphasic manner, initially impeded and then facilitated with increasing Zur concentration. These results challenge conventional models of protein unbinding being unimolecular processes and independent of protein concentration. The facilitated unbinding component likely occurs via a ternary complex formation mechanism. The impeded unbinding component likely results from Zur oligomerization on chromosome involving inter-protein salt-bridges. Unexpectedly, a non-repressor form of Zur is found to bind chromosome tightly, likely at non-consensus sequence sites. These unusual behaviors could provide functional advantages in Zur's facile switching between repression and derepression.
微生物利用锌感应调节剂来改变基因表达，以响应锌（一种必需的微量元素）可用性的变化。在锌充足的条件下，Fur 家族金属调节剂 Zur 以其金属化的阻遏物形式紧密结合到 Zur 盒操纵子位点的 DNA 上，抑制锌摄取转运体的转录。去阻遏作用来自于调节剂的解结合，在缺锌条件下，调节剂存在于其金属缺乏的非阻遏物形式中，与 Zur 盒没有明显的亲和力。虽然 Zur 对转录的阻遏机制已经得到了很好的研究，但 Zur 如何促进去阻遏作用的机制知之甚少。通过单分子/单细胞测量，我们发现，在活的大肠杆菌细胞中，Zur 从 DNA 上的解结合速率以一种前所未有的双相方式对 Zur 蛋白浓度敏感，随着 Zur 浓度的增加，最初受到阻碍，然后得到促进。这些结果挑战了蛋白解结合是单分子过程且与蛋白浓度无关的传统模型。促进解结合的成分可能通过三元复合物形成机制发生。阻碍解结合的成分可能是由于 Zur 在染色体上的寡聚化，涉及蛋白间的盐桥。出乎意料的是，发现一种非阻遏物形式的 Zur 紧密结合染色体，可能在非共识序列位点。这些异常行为可能为 Zur 在阻遏和去阻遏之间的快速切换提供了功能优势。
