CHROMATIN STRUCTURE AND STABILITY IN THE SOLUTION STATE

核小体阵列由核心组蛋白八聚体-DNA复合体组成 沿着DNA分子以大约200个碱基的间隔排列。它们是根本 染色体超结构的积木，以及所有人的底物 真核生物核中的功能过程；例如，转录， 复制、修复。溶液中的核小体阵列以平衡状态存在 在展开、适度折叠、广泛折叠和寡聚之间 构象状态。其中每一种状态的形成都是由 核心组蛋白N-末端，通过多种分子机制发挥作用。 将连接器组蛋白掺入核小体阵列形成染色质 阵列使平衡向广泛折叠和寡聚化方向移动 状态，并在这个过程中戏剧性地改变连接物DNA的结构 连接阵列中相邻的核小体。建议的目标 研究是为了描绘和描述核心的机制 组蛋白N末端和连接子组蛋白对染色质折叠的影响 以及真核RNA聚合酶的转录。具体地说，使用长度- 以及根据成分定义的核小体和染色质阵列 纯DNA和组蛋白成分，结合创新的技术 一种将分析超速离心、定量琼脂糖凝胶 凝胶电泳法、凝胶电泳法和电子冷冻法。 汉森建议：(1)描述选择性清除和定向清除的特征 核心组蛋白N末端乙酰化对核小体阵列折叠的影响 和齐聚，并确定N-末端是否形成同型或 彼此的异型复合体，(2)表征不同的接头 组蛋白结构域和序列亚型影响染色质折叠和连接物 DNA结构，以及(3)进行相关的体外转录研究 确定染色质折叠通过RNA抑制转录的程度 聚合酶II和III。拟议的实验是强烈的假设 驱动，并直接解决与以下方面相关的几个长期存在的范例 染色质的结构和功能。这些研究将有助于提供 为实现功能组装的长期目标奠定必要的基础 完全来自纯组蛋白和非组蛋白的体外重要遗传位点 组件。

Nucleosomal arrays consist of core histone octamer-DNA complexes spaced at ~200 bp intervals along a DNA molecule. They are the fundamental building blocks of chromosomal superstructures, and the substrates for all functional processes in the eukaryotic nucleus; e.g., transciption, replication, repair. Nucleosomal arrays in solution exist in equilibrium between unfolded, moderately folded, extensively folded and oligomerized conformational states. Formation of each of these states is mediated by the core histone N-termini, acting through multiple molecular mechanisms. Incorporation of linker histones into nucleosomal arrays to form chromatin arrays shifts the equilibrium towards the extensively folded and oligomerized states, and in the process dramatically alters the structure of the linker DNA that connects adjacent nucleosomes in the array. The objectives of the proposed research are to delineate and characterize the mechanisms by which the core histone N-termini and linker histones exert their effects on chromatin folding and transcription by eukaryotic RNA polymerases. Specifically, using length- and compositionally-defined nucleosomal and chromatin arrays assembled from pure DNA and histone components, in combination with an innovative technical approach that integrates analytical ultracentrifugation, quanititative agarose gel electrophoresis, and electrophoresis, and electron cryo-microscopy, Dr. Hansen proposes to: (1) characterize how selective removal and targeted acetylation of the core histone N-termini influence nucleosomal array folding and oligomerization, and to determine whether the N-termini form homotypic or heterotypic complexes with each other, (2) characterize how different linker histone domains and sequence isotypes influence chromatin folding and linker DNA structure, and (3) perform correlative in vitro transcription studies to determine the extent to which chromatin folding represses transcription by RNA polymerases II and III. The proposed experiments are strongly hypothesis driven, and directly address several long-standing paradigms relating to chromatin structure and function. These studies will help provide the foundation necessary to achieve the long-term goal of assembling functionally important genetic loci in vitro entirely from pure histone and non-histone components.