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。我们不研究分子群体，而是直接观察单个DNA分子上的RNA聚合酶和调节蛋白，在真实的时间内通过不同状态跟踪分子机制的进展，同时观察转录激活的程度。这种直接可视化通过我们称为CoSMoS（共定位单分子光谱学）的多波长单分子荧光方法成为可能。在本申请中，我们建议应用CoSMoS方法来阐明所选过程的动态机制，这些过程涉及在体外转录反应中使用原核和真核RNA聚合酶和调节蛋白来调节转录起始和延伸。我们的目标是：1）确定二级通道蛋白GreB和DksA如何动态调节细菌转录延伸复合物的功能; 2）阐明细菌延伸复合物负载一般延伸因子NusA和NusG并受其调节的机制;和3）在核提取物中和从纯化的组分中可视化真核RNA聚合酶II的起始，并描述持续合成能力因子的招募和保留的动态。

 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. 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 (colocalization 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 using both prokaryotic and eukaryotic RNA polymerases and regulatory proteins in transcription reactions in vitro. Our goals are: 1) determine how secondary channel proteins GreB and DksA dynamically modulate the function of bacterial transcription elongation complexes; 2) elucidate the mechanism by which bacterial elongation complexes are loaded with and regulated by general elongation factors NusA and NusG; and 3) visualize eukaryotic RNA polymerase II initiation in nuclear extracts and from purified components, and characterize the dynamics of processivity factor recruitment and retention.