Repression at a distance by the global regulator KorB of promiscuous IncP plasmids

KorB protein (358 amino acids) binds to 12 highly conserved sequences on th e RK2 genome and co-ordinates the expression of at least five operons encod ing genes for stable inheritance and plasmid transfer. KorB represses the t rfA, korA and klaA promoters where it binds 4 bp upstream of the -35 region (class I KorB operators, O-B) We show here that KorB on its own can also r epress the trbA, trbB, kfrA and kleA promoters where O-B is between 80 and 189 bp away from the transcription start point (class II operator). A C-ter minal deletion of 17 amino acids resulted in the loss of KorB's ability to repress through class II operator but not through class I operator. This de letion reduced multimerization of His(6)-tailed KorB protein in vitro and g reatly reduced binding specificity for fragments containing O-B sequences. At the trbBp region, where O(B)9 lies 189 bp upstream of the transcription start point, mutagenesis of a proposed secondary binding site overlapping t he trbBp -35 region had no effect on the ability of KorB to repress trbB, N evertheless, gel retardation analysis showed that KorB binding is promoted by sequences upstream and downstream of O(B)9 and that KorB can form higher order complexes on DNA. However, DNase I footprinting suggested that RNA p olymerase may interact directly with KorB bound at O(B)9 and implied that c ontacts between these proteins could be responsible for the action of KorB at a distance.