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

确定细胞如何调节锌和铜等过渡金属的吸收和流出是一个关键 在阐明金属稳态的细胞机制的组成部分。细菌模型系统提供了 理解金属反应基因调控的范例。在大肠在大肠杆菌中，金属调节剂ZntR感测Zn 过量并激活锌流出，而Zur感觉锌充足并抑制锌吸收，以保持这一必要的 金属在细胞中处于适当的生理水平。CueR是ZntR的同源物，感应细胞内Cu以激活 铜流出/解毒基因，以保持这种有毒金属最小。这里的长期目标是了解 可以出于预防和治疗的目的操纵细胞中的金属调节。为了实现这一目标， PI已经建立了一个国际上独特的研究计划，应用和开发先进的单- 分子单细胞方法来询问和理解细菌金属调节的机制 在体外和活细胞中，通过大量生物化学/生物物理学和蛋白质/遗传学进一步增强 工程方法，并与生物学家建立了合作关系。这项研究发现， 铜/锌响应转录调控的第一种机制，但也出现了新的问题。 这次更新的目的是继续这一计划，以及阐明机制，夫妇 CueR/ZntR对DNA机械张力的调节及Zur双相解束缚动力学机制 DNA，PI最近发现的两种新现象。本研究的前提是 （细菌）金属调节在生物学中，发现新的和广泛相关的调节机制， 结合单分子/细胞和批量测量的能力。该研究包含两个 具体目标，每个子目标：1）确定DNA-机械-张力偶联转录的机制 通过CueR/ZntR调节。这一目标将测试基于CueR/ZntR的解结合的发现的假设， 细胞中的染色体凝聚调节DNA的RNAP作用，CueR/ZntR可以控制RNAP对 DNA. 2)确定Zur与DNA的双相解结合动力学机制。这一目标将检验假设 关于Apo/holo-Zur显示双相（即，先压抑后促进） 随着细胞内蛋白质浓度的增加，DNA的解结合动力学。这项研究具有重要意义 因为它将阐明金属调节剂在调节金属流出和摄取中的新分子机制， 以及提供有关一般金属细胞生物学的基本知识，用于确定原因或 发展涉及类似调节过程的疾病预防，并帮助发展 的（生物）化学策略来操纵细菌锌/铜调节，以损害病原体的生长。这项研究是 创新，因为它应用/开发了新的单分子操作，成像和分析方法， 在转录调节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.