Analysis of the DNA binding and replication initiation capabilities of RepA protein

RTS1质粒的REPA蛋白由288个氨基酸组成，是启动RTS1-DNA复制所必需的反式作用蛋白。将突变的rePA基因repADELTAC143以自然构型与ORI(Rts1)克隆到pBR322中，可在37ﾟC转化大肠杆菌Pola菌株JG112，但频率较低。将P1质粒的REPA的3‘端半部分与REPADELTAC143融合，通过杂交REPA(RTS1：P1)-1产生含有ORI(RTS1)的pBR322嵌合质粒。最近，我们构建了另一个编码repA(ﾟ：P1)-2的杂合基因，该基因由rts1-rePA的N端114个氨基酸和P1-rePA的174个C端氨基酸组成。然而，即使当杂交的REPA基因与ORI(RTS1)处于自然构型时，REPA(RTS1：P1)-2也不激活ORI(RTS1)。这些结果表明，激活ORI(RTS1)的功能位于RTS1-REPA的N端，从N端延伸到AA145，但当N端的多肽被限制在N端到AA114时，该功能就丧失了。最近，我们构建了RTS1-REPA的启动子区域与pFGY1的β-半乳糖苷酶基因的融合，为定量表达自抑制活性提供了可能。利用该方法，我们正在分析杂合的REPA蛋白的活性。

The RepA protein of the Rts1 plasmid, consisting of 288 amino acids, is a trans-acting protein essential for initiation of Rts1-DNA replication. A mutant repA gene, repADELTAC143, carrying a deletion that removed the 143 C-terminal amino acids of RepA, could transform, but a low frequency, an Escherichia coli polA strain JG112 at 37ﾟC, when repADELTAC143 was cloned into pBR322 with ori(Rts1) in the natural configuration. A fusion of the 3'-terminal half of repA of the P1 plasmid to repADELTAC143 yielded a pBR322 chimeric plasmid that contained ori(Rts1) through hybrid repA(Rts1:P1)-1. This plasmid was maintained much more stably in JG112 at 37ﾟC.Recently, we constructed another hybrid repA gene encoding RepA (Rts1:P1)-2 that consists of the N-terminal 114 amino acids of Rts1-RepA and the 174 C-terminal amino acids of P1-RepA. RepA(Rts1:P1)-2, however, did not activate ori(Rts1) even when the hybrid repA gene was in the natural configuration with ori(Rts1). These findings suggest that the function to activate ori(Rts1) is located in the N-terminal half of Rts1-RepA spanning from the N-terminus to AA145, but the function is lost when the N-terminal polypeptide was restricted from the N-terminus to AA114.The autorepressor function of RepA was examined by galK expression system using pFD51 as a reporter plasmid. Recently, we constructed a fusion of the promoter region of Rts1-repA with beta-galactosidase gene of pFGY1, which made possible to express the autorepressor activity quantitatively. By the method, we are analyzing the activity of the hybrid RepA proteins.