STRUCTURE AND STABILIZATION OF THE BACTERIAL NUCLEOID

细菌核的结构和稳定性

• 批准号: 6289783
• 负责人: STEVEN B ZIMMERMAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289783
• 关键词: Escherichia coli; bacterial DNA; bacterial RNA; bacterial genetics; cell component structure /function; cell nucleus; nucleic acid denaturation; nucleoproteins; protein sequence; urea

The structure of the bacterial nucleoid and the forces which maintain its DNA in a highly compact yet accessible form are largely unknown. We have been systematically characterizing spermidine nucleoids isolated from E. coli under relatively non-denaturing conditions as an approach to these problems. The isolated nucleoids have multiple restraints to DNA unfolding; each restraint is characterized by a urea concentration giving 50% unfolding (Um value) and by the effects of various agents, particularly exogenous RNase. An RNase-sensitive restraint to DNA unfolding with Um=3.2 M urea and an RNase-resistant restraint with Um=1.8 M urea have been partially characterized. The RNase-sensitive restraint appears to be actual linkages between nucleoidal DNA and residual cell envelope which result from cotranslational insertion that occurred in the cells from which the nucleoids were isolated. Such linkages would have the following connections: DNA - RNA polymerase - mRNA - ribosomes - nascent polypeptide - residual cell membrane. Support for such a basis for the RNase-sensitive unfolding at 3.2 M urea comes from 1) effects on the stability of isolated nucleoids resulting from preexposures of cells to antibiotics; antibiotics which specifically inhibit protein or RNA syntheses caused large changes in Um values that were consistent with the proposed linkages; 2) destruction of one component of the linkage, the ribosomes, occurred at ca. 3 M urea in both isolated nucleoids and purified ribosome preparations; the ribosomes may be the specific target removed at this urea concentration; 3) the high sensitivity of nucleoidal mRNA to RNase; 4) retention of the RNase-sensitive unfolding at 3 M urea, despite removal of most DNA-binding proteins (except RNA polymerase) by salt extractions, indicating that the conventional model for DNA compaction by protein binding is not controlling the unfolding at 3 M urea. The other restraint studied, the RNase-resistant restraint, was destroyed by extended treatment of isolated nucleoids with muramidases, indicating a relation to residual murein. - antibiotics,DNA,Escherichia coli,nucleoids,proteins,RNA,urea

细菌类核的结构和维持其DNA处于高度紧凑但可接近的形式的力量在很大程度上是未知的。我们对从大肠杆菌中分离的亚精胺类核进行了系统的表征。大肠杆菌在相对非变性条件下作为解决这些问题的方法。分离的类核苷对DNA解折叠有多种抑制作用;每种抑制作用的特征是产生50%解折叠的尿素浓度（Um值）以及各种试剂（特别是外源RNA酶）的影响。RNA酶敏感的约束DNA展开与Um=3.2 M尿素和RNA酶抗性约束与Um=1.8 M尿素已部分表征。RNA酶敏感性抑制似乎是类核DNA和残留细胞包膜之间的实际连接，这是由分离类核的细胞中发生的共翻译插入引起的。这种连接有以下的联系：DNA-RNA聚合酶- mRNA -核糖体-新生多肽-残留的细胞膜.支持在3.2 M尿素下RNA酶敏感性解折叠的这种基础来自：1）细胞预暴露于抗生素对分离的类核稳定性的影响;特异性抑制蛋白质或RNA合成的抗生素引起Um值的巨大变化，这与所提出的连接一致; 2）连接的一个组分，核糖体，在约100 ℃时发生破坏。在分离的类核和纯化的核糖体制备物中均存在3 M尿素，核糖体可能是在此尿素浓度下去除的特异性靶点：3）类核mRNA对RNase的高敏感性; 4）尽管去除了大多数DNA结合蛋白，但在3 M尿素下保留RNA酶敏感性解折叠（除了RNA聚合酶），表明通过蛋白质结合的DNA压缩的常规模型在3 M尿素下不控制解折叠。研究的其他约束，RNase抗性约束，被破坏的分离的类核苷酸与溶菌酶的延长治疗，表明与残留的胞壁蛋白。- 抗生素，DNA，大肠杆菌，类核，蛋白质，RNA，尿素