Single-molecule visualization of transcription regulation mechanisms

描述（由申请人提供）：目前生物学的后基因组时代的一个中心问题是理解基因表达调控的化学和物理机制。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，并且是执行对多细胞生物体发育至关重要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”，允许它对环境刺激做出适当的反应。许多癌症和其他疾病是由基因调控紊乱引起的。 在这里，我们提出了一个全新的方法来研究体外基因调控的分子机制。我们将直接观察附着在单个DNA分子上的调控机制，而不是研究分子群体，在真实的时间内通过不同的状态跟踪机制的进展，同时观察转录激活的程度。我们实验室新开发的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阐明调控机制，直接分析在完整的调控复合物中的单个分子相互作用的动力学，而不是依赖于对单个蛋白质及其与DNA或RNA聚合酶的平衡相互作用的零碎研究数据的推断。我们将把这项技术应用于三个不同的系统，涉及在大肠杆菌中的转录起始和延伸的调节。选择每个系统是因为它是在原核生物和真核生物中功能相似的共同转录调节机制的原型。 这项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。这些基本知识有望有助于旨在开发抗传染病药物的科学研究。

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.