HISTONES AND NUCLEOSOMES IN ARCHAEA (ARCHAEBACTERIA)

古细菌（古细菌）中的组蛋白和核小体

• 批准号: 2192486
• 负责人: JOHN NEWTON REEVE
• 金额: $ 12.95万
• 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 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2192486
• 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（以前的古细菌）含有 具有一级序列的小DNA结合蛋白， 二级结构，与核小体核心组蛋白相同。 这些 古菌组蛋白在体内和体外结合并压缩DNA，形成 结构明显类似核小体。 这一发现，加上 随着最近的认识，所有的基本分子组成的 转录起始在真核生物和海洋生物中是保守的， （原真核生物）表明，结构，和功能的 核小体在基因组压缩和调节基因表达中， 从一个仍然保留在海洋中的更简单的系统演变而来。 的 所提出的实验是为了确定这是否正确。 我们将 确定HMf A和HMf B、组蛋白A和B的3D结构， 炽热甲烷热菌及其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在哪里以及如何结合的相互作用， 将DNA分子限制到NLS中将通过定点 诱变和通过分离，选择和表征 优选的和高亲和力的DNA结合位点。 核小体 在染色质内特异性定位和重新定位以调节 真核基因在转录起始水平的表达，以及 我们将确定古细菌NLS是否也是这种情况。 我们有 建立了开启和关闭四个非常强大的古细菌的条件 启动子在体内，以及程序，以分离NLS组装和交叉- 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 这些启动子指导转录起始， 启动子活性和失活，通过鉴定核酸酶保护位点 通过探针杂交、间接末端标记和引物延伸 程序. 确定基因表达信号是如何被获取的， 从染色质内激活的蛋白质仍然是 生物医学研究中的问题，也是最难解决的问题之一， 实验性的 所提出的实验不仅将确定， 组蛋白、核小体和染色质起源于大肠杆菌， 这个更简单的系统在多大程度上为研究提供了一个有效的模型 核小体的结构，核小体的定位， 核小体调节特定基因表达的机制。