MULTIFUNCTIONAL REPRESSOR KORB CAN BLOCK TRANSCRIPTION BY PREVENTING ISOMERIZATION OF RNA-POLYMERASE PROMOTER COMPLEXES

The KorB protein of broad-host-range plasmid RK2 is a transcriptional repressor involved in the control of genes for plasmid replication, conjugative transfer and stable maintenance. We have purified this protein close to homogeneity from cells harbouring an overexpression vector with the korB gene under the control of the tac promoter. KorB binds to restriction fragments bearing its proposed operator sequence, O(B). Its interaction with this palindromic site was confirmed by DNaseI or hydroxyl radical footprinting at two O(B) sequences from RK2. Comparisons showed that the O(B) context affects the nature of the footprint. Our evidence suggests that KorB is a tetramer. As such, it may be able to bind two sites simultaneously on the same or on different DNA molecules. Using the korABF promoter, which is subject to KorB repression, we demonstrate by footprinting and restriction protection that KorB and RNA polymerase can bind simultaneously. Permanganate footprinting showed that KorB represses this promoter by preventing isomerization of the RNA polymerase-promoter complex from the closed to open form.