Purification of KorA Protein from Broad Host Range Plasmid RK2: Definition of a Hierarchy of KorA Operators

Abstract

The KorA protein of promiscuous plasmid RK2 is encoded in the central control operon, which coordinates expression of genes for replication transfer and partitioning. KorA is known to repress transcription from seven promoters on the plasmid genome but at thetrfApromoter, for the vegetative replication genes, it also causes derepression oftrbAp, the first promoter for the operon whose genes are required primarily for mating pair formation prior to conjugative transfer. We have overproduced and purified KorA (101 amino acid residues). Crosslinking indicates that it exists largely as a dimer in solution. Western blotting with specific antibodies suggests that there are approximately 4000 monomers per cell in exponential phase and about 600 in stationary phase. Footprinting confirmed the expected location of the korA operator, and indicated that korA and RNA polymerase can bind simultaneously in promoter regions. Gel retardation experiments with DNA fragments, carrying each of the seven KorA operators revealed that there is a hierarchy of binding affinities. Highest affinity (Kapp= 12 to 20 nM) occurs with operators containing the 12 bp inverted repeat GTTTAGCTAAAC (klaAp, korAp and trfAp), while lower affinities (Kapp= 136 to 272 nM) occur with less perfect repeats (klcA,kleA, kleC, kfrA). In addition, specific DNA sequences flanking the 12 bp are necessary for the characteristic type I KorA binding affinity. This hierarchy may be important in providing a graded response in expression of the operons controlled by KorA as its concentration varies, as for example on transition from exponential to stationary phase.