SINGLE-MOLECULE FLUORESCENCE ANALYSIS OF TRANSCRIPTION

转录的单分子荧光分析

• 批准号: 8171039
• 负责人: SHIMON WEISS
• 金额: $ 0.3万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171039
• 关键词: Behavior; Biochemical; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Repair; DNA biosynthesis; DNA-Directed RNA Polymerase; Defect; Detection; Development; Ensure; Equilibrium; Escherichia coli; Family; Fluorescence; Functional RNA; Funding; Gene Expression; Generations; Genetic; Genetic Recombination; Genetic Transcription; Grant; Heterogeneity; Individual; Institution; Label; Lasers; Life; Malignant Neoplasms; Methods; Modeling; Molecular; Monitor; Nucleoproteins; Pathway interactions; Polymerase; Process; RNA; RNA Processing; Research; Research Personnel; Resources; Site; Source; Structure; Suppressor Genes; Time; Transcriptional Regulation; Translations; Tumor Suppressor Genes; United States National Institutes of Health; Work; human disease; insight; member; movie; promoter; single molecule; single-molecule FRET; tool; transcription factor

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 大肠杆菌RNA聚合酶（RNAP）的转录是多基金RNAP家族的良好特征成员，涉及研究静态结构或分子结合体的方法无法访问的几个机械步骤。为了了解转录机制，有必要沿着转录途径揭示和分析动态，瞬时和非平衡步骤。单分子检测（SMD）是一组新的工具，可以通过监视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），并结合了交替激发激发，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通过分析转录复合物中特征较差的过渡来理解转录；这些转变中的几个对于转录调节非常重要，因为它们构成了转录因子控制基因表达的步骤。我们建议专注于多步过渡：从RNA聚合酶到形成RNA聚合酶启动器开放式复合物的路径上发生的过渡，这些过渡发生在从RNA聚合酶启动络合物开放式复合物到初始转录复合物的路径上发生，以及从初始转录复合物转移到初始转录复合物到Mate Complectes to a Maturection eloge elonemation。拟议工作的结果将允许直接观察转录复合物的结构和机械异质性。在经过数十年的遗传，生化和结构分析后，验证或反驳未经实验验证的转录模型。并将允许生成在DNA上运行的单个功能性RNAP分子的实时分子“电影”。 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.所提出的方法适用于对DNA复制，DNA重组，DNA修复，RNA加工和RNA翻译中存在的核蛋白络合物的分析，并且当与位点特异性标记的进步结合使用时，将允许对活细胞中此类过程进行研究。