HISTONES AND NUCLEOSOMES IN ARCHAEA (ARCHAEBACTERIA)

古细菌（古细菌）中的组蛋白和核小体

• 批准号: 2519044
• 负责人: JOHN NEWTON REEVE
• 金额: $ 13.82万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2519044
• 关键词: Archaea; DNA footprinting; biochemical evolution; chromatin; circular DNA; crosslink; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; histones; microorganism genetics; molecular cloning; nucleic acid chemical synthesis; nucleic acid sequence; nucleic acid structure; nucleosomes; protein structure function; recombinant proteins; site directed mutagenesis; stoichiometry

We have discovered that the Archaea (formerly Archaebacteria) contain small DNA binding proteins that have primary sequences, and predicted secondary structures, in common with the nucleosome core histones. These archaeal histones bind and compact DNA in vivo and in vitro into structures which visibly resemble nucleosomes. This discovery, coupled with the recent recognition that all the basic molecular components of transcription initiation are conserved in the Archaea and Eukarya (formerly Eukaryotes) suggests that the structure, and the functions of nucleosomes in genome compaction and in regulating gene expression, have evolved from a simpler system that is still retained in the Archaea. The experiments proposed are to determine if this is correct. We will determine the 3D structure of HMfA and HMfB, histones A and B from Methanothermus fervidus, and the molecular stoichiometry and architecture of the nucleosome-like structures (NLS) that they form. The protein-DNA interactions that determine where and how these archaeal histones bind and constrain DNA molecules into NLS will be determined by site-directed mutagenesis and by the isolation, selection and characterization of preferred and high affinity DNA binding sites. Nucleosomes are specifically positioned and re-positioned within chromatin to regulate eukaryotic gene expression at the level of transcription initiation, and we will determine if this is also the case for the archaeal NLS. We have established conditions that turn on and off four very strong archaeal promoters in vivo, and also procedures to isolate NLS assembled and cross- linked in situ in these cells under these different conditions. We will determine where NLS are assembled in vivo, in relationship to the sites at which these promoters direct transcription initiation, under conditions of promoter activity and inactivity, by identifying nuclease protected sites by probe hybridizations, indirect end-labeling and primer extension procedures. Determining how gene expression signals are accessed and activated from within chromatin remains one of the most important questions in biomedical research, and one of the most difficult to address experimentally. The experiments proposed will not only establish if histones, nucleosomes and chromatin originated in the Archaea, but also the extent to which this simpler system provides a valid model for studies of the structure of the nucleosome, of nucleosome positioning, and of the mechanisms by which nucleosomes regulate specific gene expression.

我们发现古细菌（以前称为古细菌）含有 具有一级序列的小 DNA 结合蛋白，并预测 二级结构，与核小体核心组蛋白相同。 这些 古细菌组蛋白在体内和体外结合并压缩 DNA 结构明显类似于核小体。 这一发现，加上 最近人们认识到，所有基本分子成分 转录起始在古细菌和真核生物中是保守的 （以前的真核生物）表明，结构和功能 核小体在基因组压缩和调节基因表达中具有 从古细菌中仍保留的更简单的系统演变而来。 这 提出的实验是为了确定这是否正确。 我们将 确定 HMfA 和 HMfB、组蛋白 A 和 B 的 3D 结构 产热甲烷菌，以及分子化学计量和结构 它们形成的核小体样结构（NLS）。 蛋白质-DNA 决定这些古细菌组蛋白结合的位置和方式的相互作用 将 DNA 分子限制为 NLS 将由定点确定 诱变以及通过分离、选择和表征 优选且高亲和力的 DNA 结合位点。 核小体是 在染色质内专门定位和重新定位以调节 转录起始水平的真核基因表达，以及 我们将确定古菌 NLS 是否也是如此。 我们有 开启和关闭四种非常强大的古菌的既定条件 体内启动子，以及分离 NLS 组装和交叉的程序 在这些不同的条件下在这些细胞中原位连接。 我们将 确定 NLS 在体内组装的位置，与以下位点的关系 这些启动子在以下条件下指导转录起始 通过识别核酸酶保护位点启动子活性和失活 通过探针杂交、间接末端标记和引物延伸 程序。 确定如何获取基因表达信号并 从染色质内部激活仍然是最重要的之一 生物医学研究中的问题，也是最难解决的问题之一 实验性地。 所提出的实验不仅将确定是否 组蛋白、核小体和染色质起源于古细菌，但也 这个更简单的系统在多大程度上为研究提供了有效的模型 核小体的结构、核小体的定位以及 核小体调节特定基因表达的机制。