Unusual mechanisms of metal regulation down to single-cell single-molecule level

定义细胞如何调节过渡金属（例如锌和铜）的摄取和流出是关键 阐明金属稳态的细胞机制的组成部分。细菌模型系统提供 理解金属响应基因调控的范例。在大肠杆菌中，金属调节剂 ZntR 感应 Zn 过量并激活锌外流，而 Zur 感知锌充足并抑制锌吸收，以保持这一必需品 细胞中适当生理水平的金属。 CueR 是 ZntR 的同源物，可感知细胞内的 Cu 以激活 铜流出/解毒基因可将这种有毒金属保持在最低限度。这里的长期目标是了解如何 可以出于预防和治疗目的操纵细胞中的金属调节。为实现这一目标， PI建立了一个国际独特的研究项目，应用和开发先进的单 分子单细胞方法询问和了解细菌金属调节机制 在体外和活细胞中，通过批量生化/生物物理和蛋白质/遗传进一步增强 工程方法并与生物学家建立了合作。该研究导致了以下发现 铜/锌响应转录调控的首创机制，但也出现了新的问题。 此次更新的目的是继续该计划，并阐明耦合机制 CueR/ZntR 对 DNA 机械张力的调节以及 Zur 的双相解结合动力学机制 DNA，PI 最近发现的两种新现象。这项研究的前提包括重要性 生物学中（细菌）金属调节的研究，发现的新颖且广泛相关的调节机制，以及 结合单分子/细胞和批量测量的力量。拟议的研究包含两个 具体目标，每个目标都有子目标：1）确定DNA-机械张力-耦合转录的机制 由 CueR/ZntR 调节。该目标将测试基于 CueR/ZntR 解除绑定的发现的假设 DNA 中的 RNA 受细胞中染色体浓缩的调节，CueR/ZntR 可以控制 RNAP 的作用 脱氧核糖核酸。 2) 确定 Zur 从 DNA 中双相解离动力学的机制。这个目标将检验假设 关于 apo/holo-Zur 显示双相的初步结果（即先抑制后促进） 随着细胞内蛋白质浓度的增加，DNA 的解离动力学。研究意义重大 因为它将阐明金属调节剂在调节金属流出和吸收方面的新分子机制， 以及提供有关金属细胞生物学的基础知识，以识别原因或 制定涉及类似监管过程的疾病预防措施，并帮助发展 操纵细菌锌/铜调节以损害病原体生长的（生物）化学策略。该研究是 创新，因为它应用/开发了新颖的单分子操纵、成像和分析方法，以及 在转录调控中引入新的机械概念。

Defining how cells regulate the uptake and efflux of transition metals such as Zn and Cu is a key component in elucidating cellular mechanisms of metal homeostasis. Bacterial model systems provide paradigms for understanding metal-responsive gene regulation. In E. coli, the metalloregulator ZntR senses Zn excess and activates Zn efflux, while Zur senses Zn sufficiency and represses Zn uptake, to keep this essential metal at appropriate physiological levels in the cell. CueR, a homolog of ZntR, senses intracellular Cu to activate Cu efflux/detoxification genes to keep this toxic metal minimal. The long-term goal here is to understand how metal regulation in the cell can be manipulated for preventive and therapeutic purposes. Toward this goal, the PI has established an internationally unique research program that applies and develops advanced single- molecule single-cell approaches to interrogate and understand the mechanisms of bacterial metal regulation both in vitro and in live cells, which are further enhanced by bulk biochemical/biophysical and protein/genetic engineering approaches and established collaborations with biologists. The research has led to discoveries of first-of-their-kind mechanisms of Cu/Zn-responsive transcriptional regulation, but new questions also emerged. The objective of this renewal is to continue this program, as well as elucidate the mechanism that couples CueR/ZntR regulation to DNA mechanical tension and the mechanism of Zur’s biphasic unbinding kinetics from DNA, two novel phenomena the PI recently discovered. The premise of this research comprises the importance of (bacterial) metal regulation in biology, the discovered novel and broadly relevant regulation mechanisms, and the power of combining single-molecule/cell and bulk measurements. The proposed research contains two specific aims, each with sub-aims: 1) Identify the mechanism of DNA-mechanical-tensioncoupled transcription regulation by CueR/ZntR. This aim will test hypotheses based on the discoveries that CueR/ZntR’s unbinding from DNA is modulated by chromosome condensation in cells and that CueR/ZntR can control RNAP actions on DNA. 2) Identify the mechanism of biphasic unbinding kinetics of Zur from DNA. This aim will test hypotheses regarding the preliminary results that apo/holo-Zur shows biphasic (i.e., repressed followed by facilitated) unbinding kinetics from DNA with increasing intracellular protein concentrations. The research is significant because it will elucidate novel molecular mechanisms of metalloregulators in regulating metal efflux and uptake, as well as provide fundamental knowledge about cell biology of metals in general, for identifying causes or developing preventions of diseases that involve similar regulation processes, and for helping the development of (bio)chemical strategies to manipulate bacterial Zn/Cu regulation to impair pathogen growth. The research is innovative because it applies/develops novel single-molecule manipulation, imaging, and analysis methods, and introduce new mechanistic concepts in transcription regulation.