INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS

大肠杆菌类核的结构在很大程度上是未知的。 为了解决这个长期存在的问题，我们广泛地 其特征在于从E.杆菌 在相对非变性条件下（亚精胺类核， Kornberg等人） 分离的类核被大大稳定， 大分子拥挤，与早期的建议一致， 高度浓缩的周围细菌的体内压缩效应 细胞质（印刷中的手稿） 一种相对简单的 蛋白质从分离的类核中释放出来， 胰腺DNA酶。 三种小的DNA结合蛋白（HU、H-NS和Fis） 和来自细胞裂解过程的残留溶菌酶占 蛋白质释放，RNA聚合酶占大部分的蛋白质释放， 余项 蛋白质已通过电泳鉴定 标准，并且在H-NS和Fis的情况下，通过其N-末端氨基 酸性序列 用高盐提取分离的类核苷酸 在拥挤、稳定的条件下， 两个突出的蛋白质（HU和溶菌酶），但紧凑的形式 保留了类核物质。 这些提取的类核物质继续 在重新隔离到最初不拥挤的环境中时， 低盐培养基，表明HU，最常见的“组蛋白样” E.大肠杆菌，不是维持 在这些准备工作中。 分离的类核的暴露 到中间浓度的尿素或NaCl导致解折叠 在光学显微镜下可以看到，DNA被切割成长条状。 的DNA 在沉降性能上经历特征性变化， 或NaCl剥离了越来越多的类核蛋白。 那里 相对于NaCl的浓度， 需要去除几种蛋白质，特别是Fis和H-NS， 这可能表明存在一种盐不稳定结构， 这些蛋白质。 我们目前的重点是验证和定义这一点 结构，并将其与简单地由于 这些蛋白质和DNA之间的亲和力。

The structure of the nucleoid of Escherichia coli is largely unknown. As an approach to this long-standing problem, we have extensively characterized nucleoid preparations that were isolated from E. coli under relatively non-denaturing conditions (spermidine nucleoids, Kornberg et al.) The isolated nucleoids were greatly stabilized by macromolecular crowding, consistent with earlier suggestions of an in vivo compacting effect of the highly concentrated, surrounding bacterial cytoplasm (manuscripts in press). A relatively simple pattern of proteins was released from the isolated nucleoids upon digestion with pancreatic DNase. Three small, DNA-binding proteins (HU, H-NS, and Fis) and residual lysozyme from the cell lysis procedure account for half of the protein released, with RNA polymerase accounting for much of the remainder. The proteins have been identified by electrophoretic criteria and, in the case of H-NS and Fis, by their N-terminal amino acid sequences. Extraction of isolated nucleoids with elevated salt concentrations under crowded, stabilizing conditions efficiently removes two of the prominent proteins (HU and lysozyme), yet the compact form of the nucleoids is retained. These extracted nucleoids continue to maintain their compact form upon reisolation into the initial uncrowded low-salt medium, indicating that HU, the most common "histone-like" protein of E. coli, is not a necessary component for maintaining compaction in these preparations. Exposure of the isolated nucleoids to intermediate concentrations of urea or NaCl results in the unfolding of the DNA into long streamers, as seen by light microscopy. The DNA undergoes characteristic changes in sedimentation properties as the urea or NaCl strips off increasing amounts of the nucleoid proteins. There is considerable hysteresis with respect to the concentrations of NaCl required for the removal of several proteins, notably Fis and H-NS, possibly indicating the existence of a salt-labile structure involving these proteins. Our current focus is in validating and defining this structure and distinguishing it from the complexes formed simply due to the affinity between these proteins and the DNA.