SINGLE-MOLECULE FLUORESCENCE ANALYSIS OF TRANSCRIPTION

转录的单分子荧光分析

基本信息

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
项目类别：
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

项目摘要

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.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目和研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。大肠杆菌 RNA 聚合酶 (RNAP) 是多亚基 RNAP 家族中一个已被充分表征的成员，其转录涉及几个静态结构或分子集合体研究方法无法实现的机械步骤。为了理解转录机制，有必要揭示和分析转录途径中的动态、瞬时和非平衡步骤。单分子检测 (SMD) 是一组新工具，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。我们开发了单分子荧光共振能量转移（smFRET）与交替激光激发相结合的技术，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转变来理解转录；其中一些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。我们建议重点关注多步骤转换：从RNA聚合酶到RNA聚合酶启动子开放复合物形成路径上发生的转换，从RNA聚合酶启动子开放复合物到初始转录复合物路径上发生的转换，以及发生在RNA聚合酶启动子开放复合物路径上的转换。从最初的转录复合体到成熟的延伸复合体的路径。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；验证或反驳经过数十年未经实验验证的转录遗传、生化和结构分析后提出的模型；并将允许生成对 DNA 进行操作的单个功能性 RNAP 分子的实时分子“电影”。大肠杆菌 RNAP 聚合酶与其真核对应物的高度同源性确保了从拟议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状况。所提出的方法适用于分析 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，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此类过程。