Multifunctional repressor KorB can block transcription by preventing isomerization of RNA polymerase – promoter complexes

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Abstract
The KorB protein of broad-host-range plasmld RK2 is a transcriptlonal repressor involved In the control of genes for plasmld replication, conjugatlve 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, OB. Its Interaction with this pallndromlc site was confirmed by DNasel or hydroxyl radical footprintlng at two OB sequences from RK2. Comparisons showed that the OB 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 footprintlng and restriction protection that KorB and RNA polymerase can bind simultaneously. Permanganate footprintlng showed that KorB represses this promoter by preventing isomerization of the RNA polymerase - promoter complex from the closed to open form.