Saturation mutagenesis of the Tn10-encoded tet operator O 1: Identification of base-pairs involved in Tet repressor recognition

Abstract

Saturation mutagenesis of Tn10-encoded tet operator O 1 was performed by chemical synthesis of 30 sequence variants yielding all possible point mutations of an operator half side. Their effect on Tet repressor binding was scored by an in-vivo repressor titration system. Tet repressor affinities of selected operator mutants were further characterized in vitro by dissociation rate measurements. The O 1 sequence spans 19 base-pairs. Out of these, all 18 palindromic base-pairs are involved in Tet repressor recognition. The central base-pair does not contribute to sequence-specific binding of Tet repressor. At position 1 a pyrimidine residue is sufficient for maximal affinity to the repressor. At positions 2, 3 and 4, each mutation reduces repressor binding at least tenfold. Mutations at positions 5, 6, 7, 8 and 9 result in less drastic reductions of Tet repressor binding. Differential effects of mutations at a given position are used to deduce the chemical functions contacted by Tet repressor. The T·A to A·T transversion at position 9 increases Tet repressor affinity slightly, while all other mutations decrease repressor binding. The increased affinity of the wild-type tet operator O 2 compared to wild-type O 1 results from the addition of two favorable transversions at positions ± 9 and an unfavorable T·A to C·G transition at position −7. Deletion or palindromic doubling of the central base-pair of the O 1 palindrome reveals that the wild-type spacing of both operator half sides is crucial for efficient Tet repressor binding.