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The effect of single base pair alterations of the Tn10 encoded tet operator on recognition of Tet repressor was studied in vivo using a repressor titration system and in vitro by dissociation rate determinations of the respective complexes. Both methods reveal that the two operators, O1 and O2, which are in a tandem arrangement in the wild type, are recognized with a two-fold different affinity when separated. Studies on synthetic operator sequences indicate that the Tet repressor binds with higher affinity to the non-palindromic O2 wildtype than to the respective palindromic sequences. The in vivo repressor titration system links the expression of lacZ to the affinity of tet operator to Tet repressor. It was used to isolate tet operator mutations with reduced affinity to the repressor. The in vivo and in vitro obtained results with these mutants agree quantitatively and indicate, that the GC base pairs at positions 2, 6, and 8 are involved in interaction with the Tet repressor. Their importance for recognition decreases in that order. Transitions at position 7 of the tet operator show smaller effects on recognition than transversions.

Tn10 tetoperator mutations affecting Tet repressor recognition