CHROMATIN STRUCTURE AND STABILITY IN THE SOLUTION STATE

真核生物的DNA与组蛋白络合形成染色质。那里 是染色质的许多结构状态，以重复排列开始 核小体的核心，以中期染色体结束。而当 染色质在核调控中的基础生物学作用 过程被很好地记录下来，对分子方面的了解很少 关于染色质的任何结构的水平。这反过来又产生了 妨碍了对染色质之间的关系的清楚理解 结构和核功能。 这项提案中概述的研究克服了以前的 技术限制，并将允许在法定时间内 溶液中染色质的结构和稳定性的剖析 州政府。研究将利用一种新的体外重组染色质 由定位的核小体阵列组成的模型系统。具体的 拟议研究的目的是：(1)确定分子 最近发现的核小体阵列的两个特征的机制， 依赖于盐的折叠(在没有连接子组蛋白的情况下)和组蛋白 八聚体解离，以及(2)确定染色质折叠和 解离影响真核生物5S rRNA基因的转录。 染色质模型体系的折叠和解离 同时在分析性超速离心机中进行表征，使用 沉积速度和沉积平衡方法。 具体地说，拟议的研究将测试四个潜在的决定因素 染色质结构和稳定性：转录因子，扭转 约束和连接DNA超螺旋，核小体阵列密度，以及 多价阳离子。因为这些参数中的每个都与 染色质中的完整细胞核，这些研究将提供有价值的 洞察核小体阵列在模拟的In中所呈现的结构 活体环境。 此外，染色质模型系统还将用作 用于体外转录实验的底物，从而提供了独特的 分析染色质结构和功能效应的方法 转录过程的稳定性。如果这样做的具体目的是 建议，在这些研究完成后，将 可能实现该实验室的长期目标：生物物理学 一种活性类固醇激素调节基因的特性 在体外完全由纯化的成分重组而成。

DNA of eukaryotes is complexed with histones to form chromatin. There are many structural states of chromatin, beginning with repeated arrays of nucleosome cores and ending with the metaphase chromosome. While the fundamental biological roles of chromatin in the regulation of nuclear processes are well documented, very little is known at the molecular level about any of the structures of chromatin. This in turn has prevented clear understanding of the relationships between chromatin structure and nuclear functions. The research outlined in this proposal overcomes previous technical limitations, and will allow for the fiat time a systematic dissection of the structure and stability of chromatin in the solution state. Studies will utilize a novel in vitro reconstituted chromatin model system composed of arrays of positioned nucleosomes. The specific aims of the proposed research are to (1) determine the molecular mechanism of two recently discovered features of nucleosome arrays, salt-dependent folding (in the absence of linker histones) and histone octamer dissociation, and (2) to determine how chromatin folding and dissociation influence transcription of eukaryotic 5S rRNA genes. Chromatin model system folding and dissociation will be characterized simultaneously in the analytical ultracentrifuge, using sedimentation velocity and sedimentation equilibrium approaches. Specifically, the proposed research will test four potential determinants of chromatin structure and stability: transcription factors, torsional constraint and linker DNA supercoiling, nucleosome array density, and polyvalent cations. Because each of these parameters is associated with chromatin in the intact nucleus, these studies will provide valuable insight into the structure assumed by nucleosome arrays in a simulated in vivo environment. In addition, the chromatin model systems will also be used as a substrate for in vitro transcription experiments, thus providing a unique means to analyze the functional effects of chromatin structure and stability on the process of transcription. If the specific aims of this proposal are achieved, at the completion of these studies it will be possible to tackle the long term goal of this laboratory: biophysical characterization of an active steroid hormone-regulated gene that has been reconstituted in vitro entirely from purified components.