DNA STRUCTURE AND ACCESSIBILITY IN CHROMATIN

描述（改编自申请人的摘要）：每个真核细胞 必须执行特定基因表达的复杂程序。 的 许多遗传性疾病的解决将取决于 说明促成这一方案的所有因素。 最近的工作 已经证明，在某些情况下，染色质结构起着重要的作用， 在实现这种特定表达模式方面的重要作用， 染色质的结构元件很可能已经被 整合到许多转录控制机制中。 这一作用可能 是活跃的，实际上促进了转录的活动， 机械，或者它可能是更被动的，从而精确对准的 染色质中蛋白质-DNA接触与关键的顺式作用DNA元件 在启动子内是这样的，转录途径不是 受限 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接近区域。 此外，甚至可以呈现这些可访问区域中的元素 不能通过连接组蛋白的结合和 染色质的高级结构。 本提案的具体目的是制定一个详细的结构 了解核小体核心和连接组蛋白如何组织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 含有非洲爪蟾5S RNA基因的片段定位核小体。 一 一组均匀和明确定义的模型染色质复合物将被 建造的建筑将适合全面的结构分析 通过使用DNA结构的高分辨率化学探针， 功能分析通过评估各种结合 转录因子 这种方法避免了 通常与天然染色质制备相关的异质性 复杂和问题与这些大的复杂结构分析 通过传统的方法，如X射线晶体学。 功能 和核小体核心进入细胞的影响的结构程度。 染色质的可接近区域将被精确地确定， 评估由于连接体组蛋白和这些连接体组蛋白的折叠引起的效应 可访问区域。 这项调查将与拟议的 染色质在5S基因调控中的作用模型， 理解启动子中染色质结构的复杂作用 架构 拟议的工作也可能涉及长期存在的问题 关于接头DNA在高等生物中的构象和构型， 顺序染色质结构。

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.