STRUCTURE AND STABILIZATION OF THE BACTERIAL NUCLEOID

细菌类核的结构和使其DNA保持高度紧凑但易于接近的形式的力量在很大程度上是未知的。我们已经在相对非变性条件下系统地表征了从大肠杆菌中分离的亚精胺类核，作为解决这些问题的方法。分离的类核对DNA展开有多重限制；每个抑制的特点是尿素浓度达到50%展开（Um值），并受到各种药物，特别是外源性rna酶的影响。用Um=3.2 M尿素对DNA展开的rnase敏感抑制和用Um=1.8 M尿素对DNA展开的rnase抗性抑制进行了部分表征。rnase敏感抑制似乎是核DNA和残留细胞包膜之间的实际联系，这是由于在分离出类核的细胞中发生的共翻译插入造成的。这种键有以下联系：DNA - RNA聚合酶- mRNA -核糖体-新生多肽-残余细胞膜。支持在3.2 M尿素下rnase敏感展开的这种基础的是：1)细胞预先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合成的抗生素引起了与所提出的联系一致的Um值的巨大变化；2)在分离的类核和纯化的核糖体制备中，连锁的一个组成部分核糖体在约3 M尿素处发生破坏；核糖体可能是在这个尿素浓度下去除的特定靶标；3)核状mRNA对RNase的高敏感性；4)尽管通过盐萃取去除了大多数DNA结合蛋白（RNA聚合酶除外），但在3m尿素中仍保留了RNA酶敏感的展开，这表明传统的蛋白质结合DNA压实模型不能控制3m尿素的展开。研究的另一种抑制，rnase抗性抑制，被分离的类核与酶酰胺酶的长期处理所破坏，表明与残留的鼠蛋白有关。-抗生素，DNA，大肠杆菌，类核，蛋白质，RNA，尿素

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