Structure and Stabilization of the Bacterial Nucleoid

细菌核的结构和稳定性

• 批准号: 6105333
• 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/6105333
• 关键词: Escherichia coli; bacterial DNA; bacterial RNA; bacterial genetics; cell component structure /function; cell nucleus; nucleic acid denaturation; nucleoproteins; protein sequence; urea

Summary: The structure of the bacterial nucleoid and the forces which maintain its DNA in a highly compact yet accessible form are largely unknown. To approach these problems, we are systematically characterizing nucleoid preparations that are isolated from E. coli under relatively non-denaturing conditions (spermidine nucleoids, Kornberg et al.). In the current work, we use controlled nucleoid denaturation in urea solutions to help delineate some of the stabilizing factors of the nucleoid. We have devised an assay for nucleoid denaturation which can rapidly assess the fraction of DNA unfolding and that is readily applied to large numbers of samples. This "dilatancy" assay uses the pervasive tendency of denatured nucleiods to aggregate under the influence of low centrifugal forces. Nucleoid denaturation occurs in 3 M urea as a cooperative transition. Several properties of this transition indicate a major role for RNA in stabilizing the nucleoids: 1) Pretreatment of the nucleoids with low concentrations of RNase decreases the amount of urea required for half-denaturation (Um value) by 2-fold. 2) Exposure of the cells to rifampicin results in unstable nucleoids. 3) Denaturation is closely matched by RNA degradation. A set of DNA-associated proteins is released from the nucleoid during this transition. Nucleoids isolated from cells having mutations in several of these proteins (Fis, H-NS, StpA) have unchanged Um values. Two new DNA-associated proteins which might be involved in nucleoid function have been identified in E. coli. The amounts of both proteins released from the nucleoids by exogenous DNase are increased 2-fold in a Fis mutant. Partial amino acid sequences of the two proteins are compatible with two previously described "hypothetical proteins" predicted from DNA sequencing studies; one of these has been been shown to be expressed by Ryder et al. and may be an exonuclease. Um is greatly increased in nucleoids from chloramphenicol-treated cells. The components responsible for this "hyperstabilization" are currently under study.

摘要：细菌类核的结构 以及维持其DNA高度紧凑的力量 可访问的形式在很大程度上是未知的。为了解决这些问题， 我们正在系统地描述 分离自E.大肠杆菌在相对非变性条件下 （亚精胺类核，Kornberg等人）。在目前的工作中，我们 在尿素溶液中使用受控的类核变性， 描述了类核的一些稳定因子。我们有 设计了一种检测类核变性的方法， DNA解折叠的部分，这很容易应用于大的 样本数量。这种“免疫性”测定使用了普遍存在的 变性核素在下列因素影响下聚集的趋势 低离心力。 类核变性发生在3 M尿素中 作为一个合作的过渡。这种转变的几个性质 表明RNA在稳定类核中的主要作用：1） 用低浓度的RNase预处理类核 减少了半变性所需的尿素量（Um 值）的2倍。2)将细胞暴露于利福平导致 不稳定类核3)变性与RNA密切匹配 降解一组DNA相关蛋白质从细胞中释放出来， 在这个转变过程中。从具有以下的细胞分离的类核 这些蛋白质中的几种（Fis，H-NS，StpA）突变具有 Um值不变。两种新的DNA相关蛋白， 可能参与类核功能的基因已在E. 杆菌从类核释放的两种蛋白质的量， Fis突变体中外源DNA酶增加了2倍。部分 这两种蛋白质的氨基酸序列与两种蛋白质相容， 先前描述的从DNA预测的“假设蛋白质” 测序研究;其中一个已经被证明是 由Ryder等表达，并且可以是核酸外切酶。嗯是大大的 氯霉素处理细胞的类核增加。的 负责这种“超稳定”的组件目前 正在研究中。