SINGLE-MOLECULE FLUORESCENCE ANALYSIS OF TRANSCRIPTION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。大肠杆菌RNA聚合酶（RNAP）是多亚基RNAP家族的一员，它的转录涉及几个机制步骤，这些步骤是研究静态结构或分子集成的方法所无法达到的。为了理解转录机制，有必要揭示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物的实时行为来应对这一挑战。为了研究转录复合物的结构和动力学，我们开发了单分子荧光共振能量转移（smFRET）结合交替激光激发。我们建议使用这种方法通过分析转录复合物中特征不明显的转变来理解转录；其中一些转变对转录调控非常重要，因为它们形成了转录因子控制基因表达的步骤。我们建议将重点放在多步过渡上：从RNA聚合酶到RNA聚合酶启动子开放复合物形成的过渡，从RNA聚合酶启动子开放复合物到初始转录复合物的过渡，以及从初始转录复合物到成熟延伸复合物的过渡。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；验证或反驳经过几十年的遗传、生化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”，显示单个功能性RNAP分子在DNA上的作用。大肠杆菌RNAP聚合酶与其真核生物的高度同源性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转录，并将极大地增强对转录相关的人类疾病的理解，如各种形式的癌症（因为许多致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状况。所提出的方法适用于分析存在于DNA复制、DNA重组、DNA修复、RNA加工和RNA翻译中的核蛋白复合物，并且当与位点特异性标记的进展相结合时，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.