complementation of Rts1 RepA with phage p1 RepA protein

点击翻译按钮获取中文摘要

The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of replication. To study the functional domains of RepA,hybrid proteins were constructed of Rts1 RepA with the RepA initiator protein of plasmid P1 such that the N-terminal portion was from Rts1 RepA and the C-terminal portion from P1 RepA.Six hybrid protein were examined for function.The minimal region of Rts1 RepA required for the in trans activation of Rts1 origin encompassed the N-terminal 206 amino acids while only 129 N-terminal amino acids were required for binding to the Rts1 ori region in vitro. Both in vivo and in vitro studies showed that the N-terminal 257 amino acids fragment was required for autorepressor activity. These results cleary indicate that the N-terminal region of the RepA molecule of Rts1 is involved in the activation of the replication origin of Rts1.Among the hybrid proteins, RepAX15 consisting of the N-terminal 145 amino acids of Rts1 RepA and 142 amino acids from P1 RepA is most interesting, since it showed strong interference with both Rts1 and P1 replication, although the protein could not bind in vitro to P1 ori efficiently. It should be also stressed that no hybrid RepA activated P1 ori. These results suggest that the C-terminal region of both Rts1 and P1 RepA is involved in protein-protein interaction, perhaps in dimer formation.