DNA STRUCTURE AND ACCESSIBILITY IN CHROMATIN

描述(摘自申请者摘要)：每个真核细胞 必须执行特定基因表达的复杂程序。这个 许多基于基因的疾病的解决将取决于 对影响该计划的所有因素进行说明。近期工作 已经证明，在某些情况下，染色质结构发挥着 在形成这种特定的表达方式和 染色质的结构元素很可能是 整合到许多转录控制机制中。此角色可能 保持活跃，实际上促进了转录的活动 机械，或者它可能更被动，从而精确地对齐 染色质中的蛋白质-DNA与关键的顺式作用DNA元件的接触 启动子内部是这样的，转录途径不是 受阻了。大多数DNA结合转录因子将不能 访问这些关键元素，除非它们位于 核小体核心结构之间的染色质可及区域。 此外，甚至可以呈现这些可访问区域中的元素 不能通过连接子组蛋白的结合和形成 染色质的高阶结构。 这项建议的具体目标是制定一项详细的结构 了解核小体核心和连接子组蛋白如何组织DNA 并调节反式作用因子对DNA的可及性 染色质。这将通过利用DNA的能力来实现 包含非洲爪哇5S RNA基因的片段，用于定位核小体。一个 一组均一的和定义明确的模型染色质复合体将是 可进行全面结构分析的建筑 通过使用高分辨率的DNA结构和化学探针 功能分析通过评估各种分子的结合来实现 转录因子。这种方法回避了以下问题 异质性通常与天然染色质的制备有关 络合物及这些大型络合物的结构分析问题 通过X射线结晶学等传统方法。功能界别 以及核小体核心进入核小体的影响程度 染色质的可及区域将被精确地确定以及 连接子组蛋白的作用及其折叠的评估 可访问的区域。这项调查将与提议的 染色质在5S基因调控中的作用及一般模型 了解染色质结构在启动子中的复杂作用 建筑。拟议的工作也可能涉及长期存在的问题 关于高等植物连接体DNA的构象和构象 对染色质结构进行排序。

DESCRIPTION (Adapted from Applicant's Abstract): Each eukaryotic cell must execute a complex program of specific gene expression. The resolution of many genetically-based diseases will depend upon the elucidation of all factors contributing to this program. Recent work has demonstrate that, in some instances, chromatin structure plays an important role in bringing about this pattern of specific expression and it is likely that the structural elements of chromatin have been integrated into many transcriptional control mechanisms. This role may be active, actually facilitating the activity of the transcriptional machinery, or it may be more passive, whereby the precise alignment of protein-DNA contacts in chromatin with critical cis-acting DNA elements within promoters is such that pathways to transcription are not hindered. Most DNA-binding transcription factors will not be able to access these critical elements unless they are situated in the accessible regions of chromatin between nucleosome core structures. Further, even elements in these accessible regions may be rendered inaccessible by the binding of linker histones and the formation of higher order structures of chromatin. The specific aim of this proposal is to develop a detailed structural understanding of how nucleosome cores and linker histones organize DNA and modulate the accessibility of trans-acting factors to DNA in chromatin. This will be accomplished by exploiting the ability of a DNA fragment containing a Xenopus 5S RNA gene to position nucleosomes. A set of homogeneous and well- defined model chromatin complexes will be constructed which will be amenable to comprehensive structural analyses by the use of high resolution chemical probes of DNA structure and functional analysis by assessment of the binding of various transcription factors. This approach circumvents the problem of heterogeneity usually associated with preparation of native chromatin complexes and problems with structural analysis of these large complexes by traditional methods such as X-ray crystallography. The functional and structural extent of influence of the nucleosome core into the accessible regions of chromatin will be precisely determined as well as assessment of effects due to linker histones and folding of these accessible regions. This investigation will be relevant to proposed models for the role of chromatin in 5S gene regulation and to general understanding of the complex role of chromatin structure in promoter architecture. The proposed work may also bear on long-standing issues concerning the conformation and configuration of linker DNA in higher- order chromatin structures.