Newman G, Crooke E
Department of Biochemistry, Georgetown University Medical Center, Washington, D.C. 20007, USA.
J Bacteriol. 2000 May;182(9):2604-10. doi: 10.1128/JB.182.9.2604-2610.2000.
Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.
鉴于原核细胞中缺乏细胞核，细菌染色体复制中空间组织的重要性才刚刚开始得到充分认识。DnaA蛋白是大肠杆菌染色体复制的起始因子，它作为一种可溶性蛋白被纯化出来，并且在体外，它能在无膜的DNA合成反应中高效启动微型染色体的复制。然而，它在体外从复制无活性形式转变为活性形式是通过与脂质双层中的酸性磷脂结合实现的。为了确定DnaA蛋白在原位是存在于细胞质中、与膜相关还是两者皆有，我们使用免疫金冷冻超薄切片电子显微镜和免疫荧光技术检测了DnaA的细胞定位。这两种方法都表明DnaA定位于细胞膜，进一步表明大肠杆菌中的染色体复制起始是一个与膜相关的事件。
