INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS

大肠杆菌的类核结构在很大程度上是未知的。

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.