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

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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.