Single-molecule visualization of transcription regulation mechanisms

转录调控机制的单分子可视化

基本信息

批准号：
7931231
负责人：
JEFF GELLES
金额：
$ 12.28万
依托单位：
BRANDEIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7931231
关键词：
Affect Bacteria Binding Binding Sites Biochemical Biology Cell Differentiation process Cell physiology Chemicals Communicable Diseases Complex DNA DNA Binding DNA-Directed RNA Polymerase Data Development Disease Dyes Enhancers Equilibrium Escherichia coli Eukaryota Fluorescence Fluorescence Microscopy Gene Expression Gene Expression Regulation Genes Genetic Transcription Genomics Human Human Biology Imagery In Vitro Individual Kinetics Knowledge Label Laboratories Malignant Neoplasms Measures Mediating Molecular Molecular Machines Monitor Normal Cell Nucleotides Organism Pathway interactions Polymerase Population Population Study Prokaryotic Cells Property Proteins Public Health Reaction Regulation Regulator Genes Repression Research Research Personnel Site Stimulus System Technology Testing Time Transcript Transcription Elongation Transcription Initiation Transcriptional Activation Transcriptional Regulation Virulence base brain cell combat genetic regulatory protein improved insight instrumentation interest novel novel strategies pathogen programs promoter prototype research study response single molecule transcription factor

项目摘要

DESCRIPTION (provided by applicant): A central concern of the present post-genomic era of biology is understanding the chemical and physical mechanisms by which gene expression is regulated. Appropriate activation and repression of particular genes is necessary for maintaining normal cell function and is required for executing the programs of cell differentiation that are essential to the development of multicellular organisms. Collectively, gene regulatory systems are the "brain" of the cell that allow it to respond appropriately to environmental stimuli. Many cancers and other diseases result from deranged gene regulation. We here propose an entirely new approach to studying the molecular mechanisms of gene regulation in vitro. Instead of studying populations of molecules, we will directly visualize the regulatory machinery attached to an isolated single DNA molecule, following the progression of the machinery through its different states in real time while simultaneously observing the extent of transcriptional activation. Such direct visualization is made possible by novel multi-wavelength single-molecule fluorescence instrumentation newly developed our laboratory. This approach will allow us for the first time to elucidate regulation mechanisms by directly analyzing the dynamics of individual molecular interactions in complete regulatory complexes, instead of relying on inferences founded on data from piecemeal studies on individual proteins and their equilibrium interactions with DNA or with RNA polymerase. We will apply this technology to three different systems involved in regulation of transcription initiation and elongation in Escherichia coli. Each system was chosen because it is a prototype for a common mechanism of transcription regulation that functions analogously in both prokaryotes and eukaryotes. The proposed research will elucidate basic mechanisms of transcription regulation. In the long term this will improve public health by improving our understanding of human biology. In addition, the proposed research will help define the molecular basis for regulatory switches that affect virulence and environmental dissemination of human pathogens. This basic knowledge is expected to aid in the scientific research aimed at development of agents to combat infectious disease.

描述（由申请人提供）：当前生物学的后基因组时代的一个中心关注点是理解基因表达调节的化学和物理机制。特定基因的适当激活和抑制是维持正常细胞功能所必需的，也是执行多细胞生物发育所必需的细胞分化程序所必需的。总的来说，基因调控系统是细胞的“大脑”，使其能够对环境刺激做出适当的反应。许多癌症和其他疾病都是由基因调控紊乱造成的。我们在这里提出了一种全新的方法来研究体外基因调控的分子机制。我们将不研究分子群，而是直接可视化附着在单个 DNA 分子上的调节机制，实时跟踪该机制在不同状态下的进展，同时观察转录激活的程度。我们实验室新开发的新型多波长单分子荧光仪器使这种直接可视化成为可能。这种方法将使我们第一次能够通过直接分析完整调控复合物中单个分子相互作用的动态来阐明调控机制，而不是依赖于对单个蛋白质及其与 DNA 或 RNA 的平衡相互作用的零碎研究数据得出的推论聚合酶。我们将把这项技术应用到大肠杆菌中参与转录起始和延伸调控的三个不同系统。选择每个系统是因为它是通用转录调控机制的原型，该机制在原核生物和真核生物中的功能类似。拟议的研究将阐明转录调控的基本机制。从长远来看，这将通过提高我们对人类生物学的理解来改善公共健康。此外，拟议的研究将有助于确定影响人类病原体毒力和环境传播的调控开关的分子基础。这些基础知识有望有助于旨在开发抗击传染病药物的科学研究。