Comparative Study of Mutant RepA Protein with Its Wild Type of Plasmid Rtsl.

我们正在研究质粒Rts 1的复制蛋白的功能，该复制蛋白在较高温度（42 ℃）下不能复制。先前分离到一个突变体RepAz 279，其表现出不能Rtsl ori-激活和与mini-Rtsl不相容。使用含有（1）Rtsl-ori和incII，（2）Rtsl-ori、incII和操纵子-启动子，（3）incI和（4）操纵子-启动子的片段，通过凝胶迁移试验测试RepAz 279的DNA结合活性。结果，突变蛋白RepAz 279在条带移动模式方面与野生型RepA蛋白没有显示任何显著差异。另外，观察到RepAz 279与RepA野生型一样弯曲Rtsl-ori片段。RepAz 279基因启动子-lac融合蛋白研究表明，RepAz 279蛋白具有与野生型相同的自阻遏活性，第279位氨基酸的改变（Arg → Gly）对RepAz 279蛋白的DNA结合活性没有显著影响。RtslRepA与P1 RepA的氨基酸序列具有高度的同源性，因此，在蛋白质组学上，RtslRepA与P1 RepA的氨基酸序列具有高度的同源性，而P1 RepA与RtslRepA的氨基酸序列具有高度的同源性。研究这些杂合蛋白质给我们带来了有趣的观察结果。含有来自P1 RepA的C-末端25个氨基酸的杂合RepA蛋白具有自阻遏活性，而含有来自P1 RepA的80个氨基酸的杂合RepA蛋白没有自阻遏活性。据报道，另一种RepA蛋白的二聚体形式具有自阻遏物活性。推测RepA蛋白C端可能对其二聚化起重要作用。因此，RepAz 279中C-末端附近的氨基酸变化可能影响RepA蛋白之间的相互作用。

We are studying about the function of the replication protein of plasmid Rtsl which is not able to replicate at higher temperature (42 C). A mutant, RepAz279, which showed inability of Rtsl ori-activation and incompatibility with mini-Rtsl was previously isolated. DNA binding activity of RepAz279 was tested with gel shift assay using fragments containing (1) Rtsl-ori and incII, (2) Rtsl-ori, incII,and operator-promoter, (3) incI,and (4) operator-promoter. As results, the mutant protein RepAz279 did not show any significant differencies from wild-type RepA protein in their patterns of band shifting. Additionally, RepAz279 was observed to bend the Rtsl-ori fragment as same as RepA wild-type. RepAzs279 protein showed autorepressor activity as same with wild type by studying with promoter-lac fusion.It was concluded that amino acid change of 279th (Arg to Gly) did not have any significant effect on DNA-binding activity of RepAz279To study the role of C-terminal of RepA protein became to be important, since the amino acid change of RepAz279 is closer to its C-terminus. To do that, some of hybrid proteins which were composed of N-terminus peptide from Rtsl RepA and C-terminus peptide from P1 RepA.Rtsl RepA and P1 RepA share high homology in its amino acid sequence. To study those hybrid protein brought us interesting observations. Hybrid RepA protein containing C-terminal 25 amino acids from P1 RepA had autorepressor activity but that containing 80 amino acids from P1 RepA did not. It is reported for another RepA protein that dimer form has the autorepressor activity. It is possible that C-terminus of RepA protein has important role for its dimerization. Therefore amino acid change near C-terminus in RepAz279 may affect the interaction between RepA proteins.