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ZntR 介导的 zntA 转录对纳米级细胞内游离锌有响应。

ZntR-mediated transcription of zntA responds to nanomolar intracellular free zinc.

机构信息

Program in Chemical Biology & Department of Chemistry, The University of Michigan, 930 N University Ave, Ann Arbor, MI 48109, USA.

出版信息

J Inorg Biochem. 2012 Jun;111:173-81. doi: 10.1016/j.jinorgbio.2012.02.008. Epub 2012 Feb 22.

DOI:10.1016/j.jinorgbio.2012.02.008
PMID:22459916
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3408962/
Abstract

In E. coli, ZitB and ZntA are important metal exporters that enhance cell viability under high environmental zinc. To understand their functions in maintaining zinc homeostasis, we applied a novel genetically-encoded fluorescent zinc sensor to monitor the intracellular free zinc changes in wild type, ∆zitB and ∆zntA E. coli cells upon sudden exposure to toxic levels of zinc ("zinc shock"). The intracellular readily exchangeable zinc concentration (or "free" zinc) increases transiently from picomolar to nanomolar levels, accelerating zinc-activated gene transcription. After zinc shock, the zitB mRNA level is constant while the zntA mRNA increases substantially in a zinc-dependent manner. In the ∆zitB E. coli strain the free zinc concentration rises more rapidly after zinc shock compared to wild type cells while a prolonged accumulation of free zinc is observed in the ∆zntA strain. Based on these results, we propose that ZitB functions as a constitutive, first-line defense against toxic zinc influx, while ZntA is up-regulated to efficiently lower the free zinc concentration. Furthermore, the ZntR-mediated transcription of zntA exhibits an apparent K(1/2) for zinc activation in the nanomolar range in vivo, significantly higher than the femtomolar affinity for zinc binding and transcription activation previously measured in vitro. A kinetically-controlled transcription model is sufficient to explain the observed regulation of intracellular free zinc concentration by ZntR and ZntA after zinc shock.

摘要

在大肠杆菌中,ZitB 和 ZntA 是重要的金属外排蛋白,可增强细胞在高环境锌下的生存能力。为了了解它们在维持锌稳态中的功能,我们应用了一种新型的遗传编码荧光锌传感器来监测野生型、∆zitB 和 ∆zntA 大肠杆菌细胞在突然暴露于有毒水平锌(“锌休克”)时细胞内游离锌的变化。细胞内可交换的锌浓度(或“游离”锌)短暂地从皮摩尔增加到纳摩尔水平,从而加速锌激活基因转录。锌休克后,zitB mRNA 水平保持不变,而 zntA mRNA 则以锌依赖的方式大量增加。在 ∆zitB 大肠杆菌菌株中,与野生型细胞相比,锌休克后游离锌浓度的上升速度更快,而在 ∆zntA 菌株中观察到游离锌的积累时间延长。基于这些结果,我们提出 ZitB 作为一种组成型的、第一道防线来抵抗有毒锌的流入,而 ZntA 则被上调以有效地降低游离锌浓度。此外,ZntR 介导的 zntA 转录在体内表现出明显的锌激活 K(1/2)在纳米摩尔范围内,明显高于先前在体外测量的锌结合和转录激活的皮摩尔亲和力。动力学控制的转录模型足以解释锌休克后 ZntR 和 ZntA 对细胞内游离锌浓度的调节。

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