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

我们正在研究在较高温度（42℃）下不能复制的质粒Rtsl的复制蛋白的功能。先前已分离到一个突变体RepAz279，该突变体无法激活Rtsl，且与mini-Rtsl不相容。采用凝胶移位法检测RepAz279的DNA结合活性，使用含有(1)Rtsl-ori和incII、(2)Rtsl-ori、incII和操作子启动子、(3)incI和(4)操作子启动子的片段。结果表明，突变蛋白RepAz279与野生型RepA蛋白在条带移位模式上没有显著差异。此外，观察到RepAz279与野生型RepA一样弯曲Rtsl-ori片段。通过启动子-lac融合研究，发现RepAzs279蛋白具有与野生型相同的自抑制活性。结果表明，第279位（Arg到Gly）的氨基酸变化对RepAz279的dna结合活性没有显著影响。由于RepAz279的氨基酸变化更接近其c端，因此研究RepA蛋白c端的作用变得很重要。为了达到这一目的，一些由Rtsl RepA的n端肽和P1 RepA的c端肽组成的杂交蛋白。Rtsl RepA与P1 RepA在氨基酸序列上具有高度同源性。研究这些杂交蛋白给我们带来了有趣的观察结果。含有P1 RepA c端25个氨基酸的杂合RepA蛋白具有自抑制活性，而含有P1 RepA 80个氨基酸的杂合RepA蛋白不具有自抑制活性。据报道，另一种二聚体形式的RepA蛋白具有自抑制活性。RepA蛋白的c端可能在其二聚化过程中起重要作用。因此，reaz279中c端附近的氨基酸变化可能会影响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.