Matsunaga F, Ishiai M, Kobayashi G, Uga H, Yura T, Wada C
Institute for Virus Research, Kyoto University, Japan.
J Mol Biol. 1997 Nov 21;274(1):27-38. doi: 10.1006/jmbi.1997.1373.
The RepE protein (251 residues, 29 kDa) of mini-F plasmid, mostly found as dimers, plays a key role in mini-F replication. Whereas monomers bind to the origin to initiate replication, dimers bind to the repE operator to repress its own transcription. Among the host factors required for mini-F replication, a set of molecular chaperones (DnaK, DnaJ and GrpE) is thought to facilitate monomerization of RepE dimers. To further understand the structural basis of functional differentiation between the two forms of RepE, we examined the region(s) critical for dimerization by isolation and characterization of RepE mutants that were defective in autogenous repressor function. Such mutations were isolated from two separate regions of RepE, the central region (residues 111 to 161) and the C-terminal region (residues 195 to 208). The central region overlapped the region where the chaperone-independent copy-up mutations were previously isolated (residues 93 to 135). Likewise the mini-F mutant plasmids, carrying the mutations in the central region, could replicate in a dnaK null mutant host. One of them, S111P (111th serine changed to proline), showed a very high origin-binding activity vis-à-vis a severely reduced operator-binding activity, much like the RepE54 (R118P) mutant previously shown to form only monomers. Gel filtration and chemical crosslinking studies with purified RepE revealed that S111P primarily formed monomers, whereas other mutant proteins formed mostly dimers. On the other hand, analysis of deletion mutants revealed that the N-terminal 42 and the C-terminal 57 residues were dispensable for dimerization. Thus, the region spanning residues 93 to 161 of RepE (including Ser111 and Arg118) appeared to be primarily involved in dimerization, contributing to the negative regulation of plasmid replication.
微小F质粒的RepE蛋白（251个氨基酸残基，29 kDa）大多以二聚体形式存在，在微小F质粒复制中起关键作用。单体结合到复制起点以启动复制，而二聚体则结合到repE操纵子以抑制其自身转录。在微小F质粒复制所需的宿主因子中，一组分子伴侣（DnaK、DnaJ和GrpE）被认为有助于RepE二聚体的单体化。为了进一步了解RepE两种形式功能差异的结构基础，我们通过分离和鉴定在自身阻遏功能上有缺陷的RepE突变体，研究了对二聚化至关重要的区域。这些突变体是从RepE的两个不同区域分离得到的，即中央区域（氨基酸残基111至161）和C末端区域（氨基酸残基195至208）。中央区域与先前分离出不依赖分子伴侣的拷贝上调突变的区域（氨基酸残基93至135）重叠。同样，携带中央区域突变的微小F突变体质粒能够在dnaK缺失突变体宿主中复制。其中一个突变体S111P（第111位丝氨酸变为脯氨酸）相对于严重降低的操纵子结合活性表现出非常高的复制起点结合活性，这与先前显示仅形成单体的RepE54（R118P）突变体非常相似。对纯化的RepE进行凝胶过滤和化学交联研究表明，S111P主要形成单体，而其他突变蛋白大多形成二聚体。另一方面，对缺失突变体的分析表明，N末端的42个氨基酸残基和C末端的57个氨基酸残基对于二聚化是可有可无的。因此，RepE中跨越氨基酸残基93至161的区域（包括Ser111和Arg118）似乎主要参与二聚化，对质粒复制起负调控作用。
