Physical Chemistry of Nucleic Acids

核酸物理化学

• 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
• 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

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 核小体代表了真核RNA聚合酶转录的机械和能量屏障。 细胞中这种屏障的动态调节是基因表达调控的主要机制。 核小体屏障的不适当调节导致许多病理状况，包括癌症。 在这里，我们将使用具有单分子荧光检测的高分辨率光镊（“fleezers”）， 表征核小体对转录动力学的调节，以及人Pol II（hPol II） 影响核小体的完整性。 我们将首先表征单个hPol II的延伸动力学。具体而言，我们将遵循 hPol II在单碱基对（bp）分辨率和± 3bp的位置准确度上的进展。我们将测量 无停顿速度、停顿概率、停顿持续时间和hPol II的回溯动力学，以及测试 这些动力学是如何被诸如力、延伸因子、 RPB 1的C-末端结构域，以及模板DNA上存在扭转约束。这 分析将导致hPol II的机械化学循环的详细描述以及它是如何调节的。 与此同时，我们将使用两种方法来表征核小体屏障的能量学和动力学特征 方法：1）从组蛋白八聚体的表面机械地解开DNA，和2） 机械地解压缩围绕八聚体的DNA链。我们将研究如何 屏障由+1核小体中出现的组蛋白变体和表观遗传修饰调节（H2A.Z， H3 K9 ac和泛素化H2 B）或基因体内部（H3 K36 me 3和H3 K79 me 3）。重要的是，我们还将 联合收割机将这些力延伸测量与荧光标记的组蛋白组分的检测相结合， 八聚体（使用新建立的“fleezers”系统），以建立发生在 在DNA的机械解包和解压缩过程中，核小体被破坏。我们寻求获得一个详细的 描述屏障的高度、深度和对称性及其通过表观遗传修饰的改变。 接下来，我们将建立核小体屏障如何改变hPol II的动力学，反过来， 用fleezers系统，转录酶对核小体完整性的影响是什么。我们将 调查如何表观遗传修饰的障碍，拓扑约束的模板， 延伸因子改变hPol II的动力学以及它们如何影响屏障的稳定性， 酶与Xavier Darzacq教授合作，我们将比较hPol II的动力学- 通过追踪串联重复序列的荧光， 的MS 2噬菌体RNA结合域。我们还将进行离体实验， 核提取物我们希望获得一个前所未有的物理/生理的定量描述， 控制基因表达的机制。 1