SINGLE-MOLECULE FLUORESCENCE ANALYSIS OF TRANSCRIPTION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 大肠杆菌RNA聚合酶(RNAP)是多亚单位RNAP家族中一个特征明确的成员，它的转录涉及几个机械步骤，这些步骤是研究静态结构或分子系综的方法所无法完成的。为了了解转录机制，有必要发现和分析转录途径上的动态、瞬时和非平衡步骤。单分子检测(SMD)是一套新的工具，可以通过监测单个转录复合体的实时行为来应对这一挑战。为了研究转录复合体的结构和动力学，我们发展了单分子荧光共振能量转移(SmFRET)和交流激光激发相结合的方法。我们建议使用这种方法通过分析转录复合体中特征不佳的转变来理解转录；其中几个转变对于转录调控极其重要，因为它们形成了转录因子控制基因表达的步骤。我们建议重点研究多步转变：从RNA聚合酶到形成RNA聚合酶-启动子开放复合体的转变，发生在从RNA聚合酶-启动子开放复合体到初始转录复合体的转变，以及发生在从初始转录复合体到成熟延伸复合体的转变。拟议工作的结果将允许直接观察转录复合体的结构和机制的异质性；验证或反驳在对转录进行了数十年的遗传、生物化学和结构分析后提出的未经实验验证的模型；并将允许生成在DNA上操作的单个功能RNAP分子的实时分子“电影”。大肠杆菌RNAP聚合酶与真核细胞的高度同源性确保了从拟议的工作中获得的机械性见解将直接外推到真核转录，并将极大地提高对转录相关人类疾病的理解，如各种形式的癌症(因为许多癌基因和肿瘤抑制基因都是转录因子)、发育缺陷和其他病理条件。所提出的方法适用于DNA复制、DNA重组、DNA修复、RNA加工和RNA翻译中存在的核蛋白复合体的分析，并与位点特异性标记的进展相结合，将使研究活细胞中的这些过程成为可能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Transcription by Escherichia coli RNA polymerase (RNAP), a well-characterized member of the multisubunit RNAP family, involves several mechanistic steps inaccessible to methods that study static structures or molecular ensembles. To understand transcription mechanisms, it is necessary to uncover and analyze dynamic, transient, and non-equilibrium steps along the transcription pathway. Single-molecule detection (SMD) is a new set of tools that can stand up to this challenge by monitoring the real-time behavior of individual transcription complexes. We have developed single-molecule Fluorescence Resonance Energy Transfer (smFRET) combined with alternating-laser excitation in order to study the structure and dynamics of transcription complexes. We propose to use this method to understand transcription by analyzing poorly-characterized transitions in transcription complexes; several of these transitions are extremely important for transcriptional regulation, since they form the steps where transcription factors control gene expression. We propose to focus on multistep transitions: the transitions occurring on the path from RNA polymerase to the formation of RNA polymerase-promoter open complex, the transitions occurring on the path from RNA polymerase-promoter open complex to initial transcribing complexes, and transitions occurring on the path from initial transcribing complexes to a mature elongation complex. The results of the proposed work will allow direct observation of structural and mechanistic heterogeneity of transcription complexes; validate or disprove models proposed after decades of genetic, biochemical, and structural analysis of transcription that were not validated experimentally; and will allow generation of real-time, molecular "movies" of individual, functional RNAP molecules operating on DNA. The high homology of E. coli RNAP polymerase with its eukaryotic counterparts ensures that mechanistic insights obtained from the proposed work will be directly extrapolated to eukaryotic transcription and will greatly enhance understanding of transcription-associated human diseases, such as various forms of cancer, (since numerous oncogenes and tumor-suppressor genes are transcription factors), developmental defects, and other pathological conditions. The proposed methods are applicable to the analysis of nucleoprotein complexes present in DNA replication, DNA recombination, DNA repair, RNA processing and RNA translation, and when combined with advances in site-specific labeling, will allow the study of such processes in living cells.