Single-molecule visualization of transcription regulation mechanisms

转录调控机制的单分子可视化

• 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
• 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

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）：当前生物学后的基因组时代的核心关注点是了解基因表达的化学和物理机制。特定基因的适当激活和抑制对于维持正常的细胞功能是必要的，对于执行细胞分化程序对于发展多细胞生物至关重要的程序是必需的。总体而言，基因调节系统是细胞的“大脑”，它使其能够对环境刺激做出适当反应。许多癌症和其他疾病是由杂乱的基因调节引起的。 我们在这里提出了一种全新的方法来研究体外基因调节的分子机制。我们将直接将附着在孤立的单个DNA分子上的调节机制直接可视化，而不是研究分子的群体，这是在机械通过其不同状态实时通过其不同状态的过程，同时观察转录激活的程度。新型的多波长单分子荧光仪器新开发了我们的实验室，使这种直接可视化成为可能。这种方法将首次通过直接分析完整调节复合物中个体分子相互作用的动态来阐明调节机制，而不是依靠从对单个蛋白质及其与DNA或与RNA聚合酶进行平衡相互作用的零碎研究中建立的推论。我们将将这项技术应用于涉及大肠杆菌转录启动和伸长率调节的三种不同系统。之所以选择每个系统，是因为它是转录调节的常见机制的原型，该机制在原核生物和真核生物中都类似。 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卫生。此外，拟议的研究将有助于定义影响人类病原体毒力和环境传播的调节开关的分子基础。预计这种基本知识将有助于旨在开发药物来打击传染病的科学研究。