Xia G, Manen D, Yu Y, Caro L
Department of Molecular Biology, University of Geneva, Switzerland.
J Bacteriol. 1993 Jul;175(13):4165-75. doi: 10.1128/jb.175.13.4165-4175.1993.
The RepA replication protein of plasmid pSC101 binds as a monomer to three repeated sequences (RS1, RS2, and RS3) in the replication origin of the plasmid to initiate duplication and binds as a dimer to two inversely repeated sequences (IR1 and IR2) in its promoter region (D. Manen, L. C. Upegui-Gonzalez, and L. Caro, Proc. Natl. Acad. Sci. USA 89:8923-8927, 1992). The binding to IR2 autoregulates repA transcription (P. Linder, G. Churchward, G. X. Xia, Y. Y. Yu, and L. Caro, J. Mol. Biol. 181:383-393, 1985). A mutation in the protein RepA(cop) that affects a single amino acid increases the plasmid copy number fourfold. In vivo experiments show that, when provided in trans under a foreign promoter, the RepA(cop) protein increases the replication of a plasmid containing the origin of replication without repA, whereas it decreases the repression of its own promoter. In vitro experiments show that the purified RepA(cop) protein binds more efficiently to the repeated sequences within the origin than does RepA and that its binding to these sequences is more specific than that of RepA. Binding to an inversely repeated sequence within the repA promoter gives opposite results: the wild-type protein binds efficiently to that sequence, whereas the mutated protein binds less efficiently and less specifically. Footprint experiments confirmed these results and, in addition, showed a difference in the pattern of protection of the inversely repeated sequences by the mutant protein. Equilibrium binding experiments showed that the formation of protein-probe complexes at increasing concentrations of protein had a sigmoidal shape for binding to RS sequences and a hyperbolic shape for binding to IR sequences. The results, together with earlier work (G.-X. Xia, D. Manen, T. Goebel, P. Linder, G. Churchward, and L. Caro, Mol. Microbiol. 5:631-640, 1991), confirm that the binding of RepA to RS sequences plays a crucial role in the regulation of plasmid replication and that its binding to IR sequences plays a role in the autoregulation of RepA expression. They also demonstrate that the two separate functions of the protein are effected by two different forms of binding to the target sites.
质粒pSC101的RepA复制蛋白以单体形式与质粒复制起点处的三个重复序列（RS1、RS2和RS3）结合以启动复制，并以二聚体形式与启动子区域中的两个反向重复序列（IR1和IR2）结合（D. 马嫩、L. C. 乌佩吉 - 冈萨雷斯和L. 卡罗，《美国国家科学院院刊》89:8923 - 8927，1992年）。与IR2的结合可自动调节repA转录（P. 林德、G. 丘奇沃德、G. X. 夏、Y. Y. 于和L. 卡罗，《分子生物学杂志》181:383 - 393，1985年）。RepA(cop)蛋白中影响单个氨基酸的突变使质粒拷贝数增加四倍。体内实验表明，当在异源启动子下反式提供时，RepA(cop)蛋白可增加不含repA的复制起点的质粒的复制，而它会降低自身启动子的抑制作用。体外实验表明，纯化的RepA(cop)蛋白比RepA更有效地与复制起点内的重复序列结合，并且其与这些序列的结合比RepA更具特异性。与repA启动子内的反向重复序列结合则产生相反的结果：野生型蛋白能有效地与该序列结合，而突变蛋白的结合效率较低且特异性较差。足迹实验证实了这些结果，此外，还显示了突变蛋白对反向重复序列的保护模式存在差异。平衡结合实验表明，随着蛋白浓度增加，蛋白 - 探针复合物的形成对于与RS序列的结合呈S形，而对于与IR序列的结合呈双曲线形。这些结果与早期研究（G. - X. 夏、D. 马嫩、T. 戈贝尔、P. 林德、G. 丘奇沃德和L. 卡罗，《分子微生物学》5:631 - 640，1991年）一起证实，RepA与RS序列的结合在质粒复制调控中起关键作用，其与IR序列的结合在RepA表达的自动调节中起作用。它们还证明，该蛋白的两种不同功能是通过与靶位点的两种不同结合形式实现的。
