SINGLE-MOLECULE FLUORESCENCE ANALYSIS OF TRANSCRIPTION

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 大肠杆菌RNA聚合酶（RNAP）是多亚基RNAP家族的一个特征性成员，它的转录涉及几个研究静态结构或分子系综的方法无法达到的机械步骤。为了理解转录机制，有必要揭示和分析动态的，瞬态的，非平衡的步骤沿着转录途径。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物的实时行为来应对这一挑战。我们已经开发了单分子荧光共振能量转移（smFRET）结合交变激光激发，以研究转录复合物的结构和动力学。我们建议使用这种方法来理解转录，通过分析转录复合物中的特征不佳的转换;这些转换中的几个对于转录调控非常重要，因为它们形成了转录因子控制基因表达的步骤。我们建议把重点放在多步转换：从RNA聚合酶的RNA聚合酶启动子开放复合物的形成的路径上发生的转换，从RNA聚合酶启动子开放复合物的路径上发生的转换到初始转录复合物，从初始转录复合物的路径上发生的转换到一个成熟的延伸复合物。拟议工作的结果将允许直接观察转录复合物的结构和机制异质性;验证或反驳几十年的遗传，生物化学和转录结构分析后提出的模型，这些模型未经实验验证;并将允许生成实时的分子“电影”的个人，功能RNAP分子对DNA的操作。E.大肠杆菌RNAP聚合酶与其真核生物对应物确保了从所提出的工作中获得的机制见解将直接外推到真核生物转录，并将极大地增强对转录相关的人类疾病的理解，例如各种形式的癌症（因为许多癌基因和肿瘤抑制基因是转录因子），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物的分析，当结合位点特异性标记的进展时，将允许在活细胞中研究这些过程。

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.