HISTONES AND NUCLEOSOMES IN ARCHAEA (ARCHAEBACTERIA)

古细菌（古细菌）中的组蛋白和核小体

• 批准号: 2907400
• 负责人: JOHN NEWTON REEVE
• 金额: $ 17.75万
• 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 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2907400
• 关键词: Archaea; X ray crystallography; biochemical evolution; chromatin; circular DNA; crystallization; dimer; histones; intermolecular interaction; microorganism genetics; molecular cloning; nucleic acid sequence; nucleosomes; protein protein interaction; protein structure function; recombinant proteins; site directed mutagenesis; species difference

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（以前是考古细菌）包含 具有主要序列的小型DNA结合蛋白，并预测 二级结构，与核小体核心组蛋白共同。 这些 古细胞蛋白在体内结合和紧凑的DNA，并在体外结合 明显类似核小体的结构。 这个发现，加上 随着最近认识到的所有基本分子成分 转录启动在古细菌和Eukarya中保存 （以前是真核生物）表明结构和功能 基因组压实和调节基因表达中的核小体， 从仍保留在古细菌中的较简单的系统中演变出来。 这 提出的实验是确定这是否正确。 我们将 确定HMFA和HMFB，组蛋白A和B的3D结构 甲烷瘤肠以及分子化学计量和结构 它们形成的核小体样结构（NLS）的 蛋白-DNA 确定这些古细胞组蛋白结合的何处以及如何结合的相互作用 将DNA分子限制为NLS将通过位置定向确定 诱变以及通过隔离，选择和表征 首选和高亲和力DNA结合位点。 核小体是 专门定位并重新定位在染色质中以调节 真核基因在转录启动水平上的表达，并且 我们将确定古细菌NLS是否也是如此。 我们有 确定的条件，打开和关闭四个非常强大的古细菌 体内的启动子，以及分离NLS组装和交叉的程序 在这些不同条件下这些细胞的原位链接。 我们将 确定与位点的关系中的NL在体内组装的位置 这些启动子在此条件下直接转录启动 通过识别受核酸酶保护位点的启动子活性和不活跃 通过探针杂交，间接终端标签和底漆扩展 程序。 确定如何访问基因表达信号并 从染色质中激活仍然是最重要的 生物医学研究中的问题，也是最难解决的问题之一 实验。 提出的实验不仅会确定是否是否 组蛋白，核小体和染色质起源于古细菌，但也起源于 该简单系统为研究提供了有效模型的程度 核小体的结构，核小体定位和 核小体调节特定基因表达的机制。