Physical Chemistry of Nucleic Acids

核酸物理化学

基本信息

批准号：
9918385
负责人：
CARLOS Jose BUSTAMANTE
金额：
$ 44.11万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-07-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9918385
关键词：
Affect Affinity Base Pairing Binding C-terminal Cell Extracts Cell Nucleus Cells Chromatin Structure Collaborations DNA DNA Polymerase II DNA-Directed RNA Polymerase Dependence Detection Diffusion Diphosphates Elongation Factor Enterobacteria phage MS2 Enzymes Epigenetic Process Euchromatin Event Fluorescence Gene Expression Gene Expression Regulation Genes Genetic Transcription Height Histones Human In Vitro Individual Label Lead Location Malignant Neoplasms Measurement Measures Mechanics Messenger RNA Modification Molecular Monitor Nature Nuclear Extract Nucleic Acids Nucleosomes Pathologic Phosphorylation Physical Chemistry Physiological Polymerase Positioning Attribute Post-Translational Protein Processing Probability Process RNA Recognition Motif Recombinant DNA Regulation Reporter Resolution Role Structure Surface System Tandem Repeat Sequences Technology Testing Torsion Transcription Elongation Variant Yeasts dimer experimental study in vivo instrument laser tweezer particle single molecule transcription factor transcription factor S-II uH2A ultra high resolution

项目摘要

Project Summary Nucleosomes represent a mechanical and energetic barrier to transcription by eukaryotic RNA polymerases. The dynamics modulation of this barrier in the cell is a major mechanism of gene expression regulation. Improper regulation of the nucleosomal barrier results in numerous pathological conditions, including cancer. Here, we will use high resolution optical tweezers with single molecule fluorescence detection (“fleezers”) to characterize the modulation of transcriptional dynamics by nucleosomes, and how the human Pol II (hPol II) affects nucleosome integrity. We will first characterize the elongation dynamics of single hPol II. Specifically, we will follow the progress of hPol II at single base pair (bp) resolution and at a position accuracy of ±3 bp. We will measure the pause-free velocity, the pausing probability, pause duration, and backtracking dynamics of hPol II, and test how these dynamics are modulated by factors such as force, elongation factors, the phosphorylation state of the C-terminal domain of RPB1, as well as the presence of torsional constrains on the template DNA. This analysis will results in a detailed description of the mechanochemical cycle of hPol II and how it is regulated. In parallel, we will characterize the energetics and dynamics of the nucleosomal barrier using two approaches: 1) mechanically unwrapping the DNA from the surface of the histone octamer and 2) mechanically unzipping the strands of the DNA sequentially around the octamer. We will investigate how the barrier is modulated by histone variants and epigenetic modifications that appear in +1 nucleosomes (H2A.Z, H3K9ac and ubiquitinated H2B) or inside gene bodies (H3K36me3 and H3K79me3). Importantly, we will also combine these force-extension measurements with detection of fluorescently labelled histone components of the octamer (using a newly built “fleezers” system) to establish the structural changes that occur in the nucleosome during mechanical unwrapping and unzipping of the DNA. We seek to obtain a detailed description of the height, depth, and symmetry of the barrier and its alteration by epigenetic modifications. Next, we will establish how the nucleosomal barrier modifies the dynamics of hPol II and, in turn, what is the effect of the transcribing enzyme on the integrity of nucleosomes using the fleezers system. We will investigate how epigenetic modifications of the barrier, the topological constraint of the template, and elongation factors alter the dynamics of hPol II and how they affect the stability of the barrier to the passage of the enzyme. In collaboration with Prof. Xavier Darzacq, we will compare the dynamics of hPol II obtained in- vitro with those observed in-vivo in the context of nucleosomes, by tracking the fluorescence of tandem repeats of MS2 bacteriophage RNA binding domains in U2OS cells. We will also perform ex-vivo experiments using nuclear extracts. We hope to obtain an unprecedented quantitative description of the physical/physiological mechanisms that control gene expression. 1

项目摘要核小体代表着真核RNA聚合酶转录的机械和能量障碍。细胞中该障碍的动力学调节是基因表达调节的主要机制。核渗透屏障的调节不当导致许多病理状况，包括癌症。在这里，我们将使用具有单分子荧光检测（“ Fleezer”）的高分辨率光学镊子表征核组对转录动力学的调节，以及人类pol II（HPOL II）影响核小体完整性。我们将首先表征单个HPOL II的伸长动力学。具体来说，我们将遵循 HPOL II在单基对（BP）分辨率和位置精度±3 bp时的进展。我们将衡量无暂停速度，暂停概率，暂停持续时间和HPOL II的回溯动力学和测试这些动力学是如何通过诸如力，伸长因子，磷酸化状态等因素调节的 RPB1的C末端结构域以及对模板DNA的扭转约束。这分析将导致对HPOL II的机械化学周期及其调节的详细描述。同时，我们将使用两个方法：1）机械地从组蛋白八聚体的表面机械解开DNA，2）机械地将DNA的链依次解开八聚体。我们将调查如何障碍物由组蛋白变体和表观遗传修饰调节，这些变体出现在+1核小体中（H2A.Z，，， H3K9AC和泛素化H2B）或基因体内（H3K36Me3和H3K79Me3）。重要的是，我们也会将这些力扩展测量与检测到荧光标记的Hisstone组件的检测 Octamer（使用新建的“ Fleezers”系统）来确定发生的结构变化 DNA的机械解开和解压缩过程中的核小体。我们寻求获得详细的描述屏障的高度，深度和对称性及其对表观遗传修饰的改变。接下来，我们将确定核宇宙屏障如何修饰HPOL II的动力学，然后又如何修改使用Fleezers系统，转录酶对核小体完整性的影响是什么？我们将研究屏障的表观遗传修饰，模板的拓扑约束以及伸长因子改变了HPOL II的动力学以及它们如何影响障碍通过的稳定性酶。与Xavier Darzacq教授合作，我们将比较HPOL II的动力学。通过跟踪串联重复的荧光，在核小体中观察到体内的体面 U2OS细胞中的MS2细菌RNA结合结构域的MS2。我们还将使用核提取物。我们希望获得对物理/生理的前所未有的定量描述控制基因表达的机制。 1