Functional domains of Rts1 RepA analyzed by hybrid proteins with P1 RepA.

1.质粒Rts 1的RepA蛋白由288个氨基酸组成，是启动质粒复制所必需的反式作用蛋白。为了研究RepA的功能结构域，构建了由286个氨基酸组成的Rts 1 RepA与原噬菌体P1 RepA的杂合蛋白，其中N端部分来自Rts 1 RepA，C端部分来自P1 RepA。发现Rts 1 RepA的N-末端区域在氨基酸残基113和129之间对于Rts 1 ori体外结合是重要的。第177 ~ 206位残基是Rts 1 RepA在体内激活Rts 1 ori的重要亚区. P1 RepA在体外与Rts 1 ori的结合强度与Rts 1 RepA一样强，在体内可激活Rts 1复制起点，但激活效率较低。相反，Rts 1 RepA在体外既不与P1 ori结合，也不激活P1 ori.通过将P1 RepA的一个小区域替换为Rts 1 RepA的相应区域，残基145-176，Rts 1 ori激活效率显著提高。发现P1 RepA的相同亚区对于P1起点的体内活化是重要的，因此，在P1 RepA分子上以及在Rts 1 RepA分子上分配了有效活化复制起点所必需的结构域。应该注意的是，该结构域不同于体外结合起点所必需的区域，尽管这两个区域都是体内激活复制起点所必需的。

1. The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA,hybrid proteins of Rts1 RepA with the prophage P1 RepA,consisting of 286 amino acids, were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA.Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. As an essential subregion of Rts1 RepA for activation of Rts1 ori in vivo, the residues between 177 and 206 was tentatively assigned.2. P1 RepA was found to bind in vitro to Rts1 ori as strongly as Rts1 RepA.In addition, P1 RepA activated in vivo the Rts1 replication origin, although the activation was quite inefficient. In contrast, Rts1 RepA showed neither binding in vitro to P1 ori nor activation in vivo of the origin.3. By replacing a small region of P1 RepA with the corresponding region of Rts1 RepA,the residues 145-176, the efficiency of Rts1 ori activation increased remarkably. The same subregion of P1 RepA was found to be important for in vivo activation of the P1 origin.Thus, a domain essential for an efficient activation of the replication origin was assigned on the P1 RepA molecule as well as on the Rts1 RepA molecule. It should be noted that the domain was distinct from a region necessary for in vitro binding to the origin, although both regions were required for in vivo activation of the replication origin.