Dissection of the switch between genes for replication and transfer of promiscuous plasmid RK2: basis of the dominance of trfAp over trbAp and specificity for KorA in controlling the switch.

The trfA and trb operons of broad host range plasmid RK2 are required for replication and conjugative transfer, respectively. Transcription of the trb operon can be initiated at one of two promoters: trbAp or trbBp. trbBp provides a burst of trb transcription on first entry into the cell. trbAp appears to be responsible for steady-state transcription of the trb operon as well as trbA, encoding a repressor which helps to shut down trbBp. The promoters trfAp and trbAp are arranged as face-to-face divergent promoters. trfAp is very strong and shuts off trbAp activity until trfAp is inhibited by KorA, one of the plasmid-encoded global regulators. Although trfAp is also repressed by KorB, a second global regulator encoded along with KorA in the central control operon, trbAp activation only occurs when KorA is present. KorB did not activate trbAp and indeed had a significant inhibitory effect on KorA activation. In vitro trfAp binds RNA polymerease (RNAP) approximately ten times more strongly than trbAp. Comparison of single and multiple rounds of in vitro run-off transcription suggested that the inhibitory effect of trfAp is due to elongating transcription complexes. In vitro studies with purified KorA and KorB on RNAP binding, isomerization and in vitro transcription suggested that both proteins can displace RNAP from trfAp, but that once open complexes have formed at either promoter they have a good chance of generating a transcript even if they encounter an opposing RNAP. In vivo KorB repressed trbAp even when trbAp was derepressed by a trfAp-1 mutation, removing the need for KorA. This suggested that KorB not only fails to derepress but actually represses trbAp despite the KorB operator being located 90 bp downstream of the transcription start point (tsp). By contrast KorA still activated trbAp when the two promoters were moved further apart or were brought so close that RNAP binding to the two promoters was mutually exclusive. Thus, KorA plays the dominant role in achieving the balance of expression of genes for alternate modes of plasmid propagation but its action is modulated by KorB.