STUDIES OF NUCLEOSOME STABILITY AND TRANSCRIPTION

生物有机体必须复杂地存储并有效地读取其DNA中包含的大量遗传信息。 参与这些过程的许多基于DNA的酶作为高度进行性的分子马达发挥作用，能够在不脱离DNA模板的情况下移位数千个碱基对。 这些马达的运动面临着机械障碍，特别是在染色质的高度包装的DNA中，并且已知这些障碍中的许多是基因表达的重要调节器。 这项提案的主要目标是解决这些基于DNA的马达如何处理这些障碍的问题。与裸DNA相比，染色质中的DNA是高度紧凑的。 核小体是染色质的主要包装单位，由大约两圈DNA包裹在核心组蛋白八聚体周围组成。RNA聚合酶在转录过程中处理核小体的分子机制在分子水平上还没有完全理解，仍然是生物学中最基本的问题之一。我们提出了一个独特的，单分子，生物物理方法来解决RNA聚合酶如何处理核小体的问题。 该方法结合了光学捕获和纳米精度位置检测技术，并补充了正在进行的生物化学和结构研究。 这种方法，这已被证明是一个强大的工具，用于研究裸DNA上的转录，将提供直接测量和可视化的个别分子事件的转录染色质在体外。 提出了两个具体目标：（1）与核小体相关的DNA的机械稳定性，以及（2）通过核小体的转录。 目的1通过从端到端拉伸核小体DNA并测量破坏核小体所需的张力来确定组蛋白-DNA相互作用的强度。 目标2通过监测转录期间RNA聚合酶的单个分子的运动并同时检测可能的核小体破坏事件，直接观察核小体在与转录RNA聚合酶相遇期间的命运。 利用这些方法，我们将确定组蛋白乙酰化和染色质重塑复合物对通过核小体DNA转录的影响。这一基础研究将有助于阐明真核生物的转录机制，并将进一步为其他基于核酸的分子马达在单分子水平上的力学研究奠定技术基础。

Biological organisms must compactly store and yet efficiently read the huge amounts of genetic information contained in their DNA. Many DNA-based enzymes involved in these processes function as highly processive molecular motors capable of translocating over thousands of base pairs without detaching from the DNA template. These motors face mechanical obstacles to their movement, especially in the highly packed DNA of chromatin, and many of these obstacles are known to be important regulators of gene expression. The broad goal of this proposal is to address the question of how these DNA-based motors deal with these obstacles. DNA in chromatin is highly compact as compared to naked DNA. The primary packing unit of chromatin, the nucleosome, consists of roughly two turns of DNA wrapped around a core histone octamer. The molecular mechanism by which RNA polymerase deals with nucleosomes during transcription is not fully understood at the molecular level and remains one of the most fundamental questions in biology. We propose a unique, single-molecule, biophysical approach to address the question of how RNA polymerase deals with nucleosomes. The proposed method combines optical trapping with nanometer-precision position detection techniques, and complements ongoing biochemical and structural studies. This approach, which has proven to be a powerful tool for the study of transcription on naked DNA, will provide direct measurements and visualization of individual molecular events of transcription in chromatin in vitro. Two specific aims are proposed: (1) mechanical stability of DNA associated with nucleosomes, and (2) transcription through nucleosomes. Aim number 1 determines the strength of histone-DNA interactions by stretching a nucleosomal DNA from end-to-end and measuring the tension required to disrupt the nucleosomes. Aim number 2 makes a direct observation of the fate of a nucleosome during an encounter with a transcribing RNA polymerase by monitoring the movement of single molecules of RNA polymerase during transcription and simultaneously detecting possible nucleosome disruption events. Using these methods, we will determine the effects of histone acetylation and chromatin remodeling complexes on transcription through nucleosomal DNA. This proposed basic research will help to elucidate the mechanisms of transcription in eukaryotes, and will further establish the technical foundation for mechanical studies of other nucleic acid-based molecular motors at the single molecule level.