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Physical Chemistry of Nucleic Acids

核酸物理化学

基本信息

批准号：
10331744
负责人：
CARLOS Jose BUSTAMANTE
金额：
$ 44.07万
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 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10331744
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 Regulation Reporter Resolution Ribosomal DNA Role 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

项目概要核小体代表真核 RNA 聚合酶转录的机械和能量屏障。细胞中这一屏障的动态调节是基因表达调控的主要机制。核小体屏障的不当调节会导致多种病理状况，包括癌症。在这里，我们将使用具有单分子荧光检测功能的高分辨率光镊（“fleezers”）来表征核小体对转录动力学的调节，以及人类 Pol II (hPol II) 如何调节影响核小体的完整性。我们将首先表征单个 hPol II 的伸长动态。具体来说，我们将遵循 hPol II 在单碱基对 (bp) 分辨率和 ±3 bp 位置精度下的进展。我们将测量 hPol II 的无暂停速度、暂停概率、暂停持续时间和回溯动态，以及测试这些动力学如何通过力、伸长因子、磷酸化状态等因素进行调节 RPB1 的 C 端结构域，以及模板 DNA 上扭转约束的存在。这分析将详细描述 hPol II 的机械化学循环及其调控方式。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方法：1) 从组蛋白八聚体表面机械地解开 DNA，2) 围绕八聚体依次机械地解开 DNA 链。我们将调查如何屏障受到+1核小体中出现的组蛋白变体和表观遗传修饰的调节（H2A.Z， H3K9ac 和泛素化 H2B）或基因体内（H3K36me3 和 H3K79me3）。重要的是，我们还将将这些力延伸测量与荧光标记的组蛋白成分的检测相结合八聚体（使用新构建的“fleezer”系统）来确定发生在 DNA 机械解包和解拉链过程中的核小体。我们寻求获得详细的屏障的高度、深度和对称性及其通过表观遗传修饰的改变的描述。接下来，我们将确定核小体屏障如何改变 hPol II 的动力学，进而，使用 flezers 系统转录酶对核小体完整性有何影响。我们将研究势垒的表观遗传修饰、模板的拓扑约束以及伸长因子改变 hPol II 的动力学以及它们如何影响屏障通过的稳定性酶。与 Xavier Darzacq 教授合作，我们将比较以下获得的 hPol II 的动态：通过追踪串联重复序列的荧光，在核小体背景下进行体外观察和体内观察 U2OS 细胞中的 MS2 噬菌体 RNA 结合域。我们还将使用以下方法进行离体实验核提取物。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

项目成果

期刊论文数量（84）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（3）

Circular dichroism studies on single Chinese hamster cells.

中国仓鼠单个细胞的圆二色性研究。

DOI：
10.1021/bi00340a600
发表时间：
1985
期刊：
Biochemistry
影响因子：
2.9
作者：
Maestre,MF;Salzman,GC;Tobey,RA;Bustamante,C
通讯作者：
Bustamante,C

Contribution of differential scattering of circularly polarized light to the optical rotatory dispersion of a sample.

圆偏振光的差异散射对样品旋光色散的贡献。

DOI：
10.1364/josaa.1.001114
发表时间：
1984
期刊：
Journal of the Optical Society of America. A, Optics and image science
影响因子：
0
作者：
Bustamante,C
通讯作者：
Bustamante,C

Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis.

脉冲场凝胶电泳过程中 DNA 分子运动的模型和计算机模拟。

DOI：
10.1021/bi00235a021
发表时间：
1991
期刊：
Biochemistry
影响因子：
2.9
作者：
Smith,SB;Heller,C;Bustamante,C
通讯作者：
Bustamante,C

Model computations on the differential scattering of circularly polarized light (CIDS) by dense macromolecular particles.

致密大分子颗粒对圆偏振光 (CIDS) 的微分散射的模型计算。

DOI：
10.1002/bip.360240505
发表时间：
1985
期刊：
Biopolymers
影响因子：
2.9
作者：
Keller,D;Bustamante,C;Maestre,MF;TinocoJr,I
通讯作者：
TinocoJr,I

A two-state kinetic model for the unfolding of single molecules by mechanical force.

通过机械力展开单分子的二态动力学模型。

DOI：
10.1073/pnas.172525099
发表时间：
2002
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Ritort,F;Bustamante,C;TinocoJr,I
通讯作者：
TinocoJr,I