Facilitated Mechanisms in Zinc Regulation down to the Single-Molecule Level

描述（由申请人提供）：锌感应调节剂使细胞能够控制锌的摄取、外排和储存，这对所有生物体的细胞活力至关重要。这些金属调节因子的许多机制途径仍然知之甚少。这里的长期目标是了解如何控制细胞中的锌稳态以达到预防和治疗目的。这里的总体目标是定义细菌锌感应金属调节因子（例如，ZntR， Zur和CzrA）如何确保在锌过量或缺乏时各自的调控在激活和失活之间以及在抑制和去抑制之间迅速切换。基于初步研究和过去的结果，中心假设是这些金属调节因子在调节转录过程中使用便利的机制，使蛋白质从DNA中解离或从全息调节因子中去除金属。为了验证这一中心假设，两个pi将合作并使用体外和体内蛋白质- dna相互作用的单分子荧光成像，蛋白质- dna相互作用动力学，体内转录谱分析，染色质免疫沉淀以及蛋白质和基因工程的组合方法。这项研究的基本原理是，了解它们的调节机制将有助于设计策略和开发药物来控制细菌锌的摄取或外排，以限制它们的生长，从而有助于预防和管理细菌感染性疾病。提出的研究有三个具体目的：(1)确定ZntR促进转录失活的机制。这里的工作假设是，锌外排的转录失活可以通过(1)辅助蛋白质解离途径发生，其中自由载子zntr，抑制因子，帮助激活因子，holo-ZntR，从DNA中解离，和/或(2)直接蛋白质替代途径，其中自由载子zntr直接替代DNA中的holo-ZntR。(2)确定Zur促进转录去抑制的机制。这里的工作假设是（#3）游离载脂蛋白zur可以帮助抑制因子holo-Zur从DNA中解离，导致Zn摄取的去抑制，（#4）杆菌硫醇（BSH）可以促进Zn从DNA结合的holo-Zur中去除，将其转化为载脂蛋白zur，后者将从DNA中解离，导致去抑制。(3)明确了CzrA促进转录去抑制和抑制的机制。这里的工作假设是（#5）自由的holo-CzrA可以帮助载脂蛋白CzrA，抑制因子，从DNA中解离，导致Zn外排的去抑制，（#6）BSH可以帮助Zn从自由的holo-CzrA中去除，并加速随后的CzrA与DNA的结合，以重建抑制。该研究具有重要意义，因为它将导致（生物）化学策略的发展，以操纵细菌锌调节来损害病原体的生长，并将填补锌调节机制的知识空白。这项研究具有创新性，因为它整合了物理和生物科学，结合了体外和体内的单分子和集合水平测量，并引入了转录调控的新概念。

DESCRIPTION (provided by applicant): Zinc-sensing regulators enable cells to control Zn uptake, efflux, and storage that are critical for cell viability across all organisms. Many mechanistic pathways for these metalloregulators are still poorly understood. The long-term goal here is to understand how Zn homeostasis in the cell can be manipulated for preventative and therapeutic purposes. The overall objective here is to define how bacterial Zn-sensing metalloregulators (e.g., ZntR, Zur, and CzrA) ensure prompt switching between activation and deactivation and between repression and de-repression of their respective regulons in response to Zn excess or deficiency. The central hypothesis, formulated based on preliminary studies and past results, is that these metalloregulators use facilitated mechanisms for either protein dissociation from DNA or metal removal from the holo regulators in regulating transcription. To test this central hypothesis, the two PIs will collaborate and use the combined approach of single-molecule fluorescence imaging of protein-DNA interactions in vitro and in vivo, ensemble protein-DNA interaction kinetics, in vivo transcription profiling, chromatin immunoprecipitation, and protein and genetic engineering. The rationale for the proposed research is that the understanding of their regulation mechanisms will help in devising strategies and developing drugs to manipulate bacterial Zn uptake or efflux to limit their growth, thus contributing to the prevention and management of bacterial infectious diseases. The proposed research has three specific aims: (1) Identify the facilitated mechanisms of transcription deactivation by ZntR. The working hypotheses here are that the transcription deactivation of Zn efflux can occur via (#1) an assisted protein dissociation pathway, where free apo-ZntR, the repressor, assists the holo-ZntR, the activator, to dissociate from DNA, and/or (#2) a direct protein substitution pathway, where free apo-ZntR directly substitutes for the holo-ZntR on DNA. (2) Identify the facilitated mechanisms of transcription de-repression by Zur. The working hypotheses here are that (#3) free apo-Zur can assist holo-Zur, the repressor, to dissociate from DNA, leading to de-repression of Zn uptake, and (#4) bacillithiol (BSH) can facilitate Zn removal from DNA-bound holo-Zur to convert it to apo-Zur, which will dissociate from DNA leading to de-repression. (3) Identify the facilitated mechanisms of transcription de-repression and repression by CzrA. The working hypotheses here are that (#5) free holo-CzrA can assist apo-CzrA, the repressor, to dissociate from DNA, leading to de-repression of Zn efflux, and (#6) BSH can assist Zn removal from free holo-CzrA and accelerate subsequent CzrA binding to DNA for reestablishing repression. The research is significant because it will lead to the development of (bio)chemical strategies to manipulate bacterial Zn regulation to impair growth of pathogens and will fill knowledge gaps in Zn regulation mechanisms. The research is innovative because it integrates physical and biological sciences, combines both single-molecule and ensemble level measurements in vitro and in vivo, and introduces new concepts in transcription regulation.