Single-molecule visualization of transcription regulation mechanisms

描述（由申请人提供）：当前后基因组时代生物学的一个中心问题是理解基因表达被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，也是执行细胞分化程序所必需的，而细胞分化是多细胞生物发育所必需的。总的来说，基因调控系统是细胞的“大脑”，它允许细胞对环境刺激做出适当的反应。许多癌症和其他疾病都是由于基因调控失常造成的。在这个研究项目中，我们已经开发并开始应用一种全新的方法来研究体外转录和转录调控的分子机制。我们不是研究分子群体，而是直接在分离的单个DNA分子上可视化RNA聚合酶和调控蛋白，实时跟踪分子机制在不同状态下的进展，同时观察转录激活的程度。这种直接可视化是通过多波长单分子荧光方法实现的，我们称之为CoSMoS（共定位单分子光谱）。在这个应用中，我们建议应用CoSMoS方法来阐明参与转录起始和延长调控的选定过程的动态机制

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 allows it to respond appropriately to environmental stimuli. Many cancers and other diseases result from deranged gene regulation. In this research project, we have developed and begun applying an entirely new approach to studying the molecular mechanisms of transcription and transcription regulation in vitro. Instead of studying populations of molecules, we directly visualize the RNA polymerase and regulatory proteins on an isolated single DNA molecule, following the progression of the molecular machinery through its different states in real time while simultaneously observing the extent of transcriptional activation. Such direct visualization is made possible by a multi-wavelength single-molecule fluorescence approach we call CoSMoS (co localization single- molecule spectroscopy). In this application, we propose applying the CoSMoS approach to elucidating the dynamic mechanisms of selected processes involved in regulation of transcription initiation and elongation in vitro using purified Escherichia coli proteins in reconstituted transcription reactions. Our goals are: 1) Test the hypothesis that RNAP holoenzyme molecules reach promoters through a bind-and-slide mechanism; 2) Define the dynamic mechanisms by which secondary channel factors act alone and together to regulate transcription initiation and elongation; and 3) Define the dynamic mechanism by which the presence of the 70 and NusA components of the transcription apparatus is remodeled in the transition between transcription initiation and elongation.