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

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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.