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摄取和外流是关键 在阐明金属动态平衡的细胞机制中的作用。细菌模型系统提供了 理解金属反应基因调控的范例。在大肠杆菌中，金属调节剂锌锌受体感应锌 过量并激活锌的外流，而Zur感觉到锌的充足并抑制锌的吸收，以保持这一必要 细胞中处于适当生理水平的金属。CueR是ZntR的同源物，它感觉细胞内的铜被激活 铜排出/解毒基因，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 细胞中的金属调节可以用于预防和治疗目的。为了实现这一目标， PI建立了国际上独一无二的应用和开发先进单项技术的研究计划。 分子单细胞方法研究和理解细菌金属调控机制 在体外和在活细胞中，通过大量的生化/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关系。这项研究导致了对 这是第一个铜/锌响应转录调控的机制，但也出现了新的问题。 这次更新的目的是继续这一计划，并阐明夫妇之间 CueR/ZntR对DNA机械张力的调节及Zur的两相解离动力学机制 DNA，这是PI最近发现的两种新现象。本研究的前提是 生物学中的(细菌)金属调节，发现的新的和广泛相关的调节机制，以及 单分子/细胞测量和整体测量相结合的能力。这项拟议的研究包括两项 特定的目标，每个都有子目标：1)识别dna-机械-张力偶联转录的机制 受CueR/ZntR的调控。这一目标将检验基于CueR/ZntR解绑的发现的假设 来自DNA的DNA是由细胞中的染色体凝聚调控的，CueR/ZntR可以控制RNAP在 DNA2)确定Zur与DNA的两相解离动力学机制。这个目标将检验假说。 关于apo/holo-zur呈双相(即先抑制后促进)的初步结果 随着细胞内蛋白质浓度的增加，解离DNA的动力学。这项研究具有重要意义 因为它将阐明金属调节剂调节金属外流和吸收的新的分子机制， 以及提供一般金属细胞生物学的基本知识，以确定原因或 开发涉及类似监管过程的疾病预防，并帮助开发 (生物)化学策略来操纵细菌的锌/铜调节，以削弱病原体的生长。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，以及 在转录调控中引入新的机械论概念。

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.