Quantitative analysis of Tn10Tet repressor binding to complete set oftetoperator mutants

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Abstract
A saturating oligonucleotide-directed mutagenesis of both tet operators in the tet regulatory sequence was performed yielding mutants with four identical base pair exchanges at equivalent positions in the four tet operator half sides. The mutants were cloned between bipolar lacZ and galK indicator genes on a multicopy plasmid allowing the quantitative analysis of their effects in vivo. In the absence of Tet repressor the mutations lead to considerably different expression levels of both genes. They are discussed with respect to the promoter consensus sequences. In particular, the -10 region of the in vivo active tetPR2 promoter is unambiguously defined by these results. In the presence of Tet repressor most of the mutants exhibit a lower affinity for that protein as determined quantitatively by their reduced expression levels. In general, tet operator recognition is most strongly affected by alterations of base pairs near the center of the palindromic sequence. The most important position is the third base pair, followed by base pairs two, four, five and six, the latter showing similar effects as base pair one. At each position, the four possible base pairs show different affinities for Tet repressor. They are discussed according to their exposure of H-bond donors and -acceptors in the major and minor grooves of the B-DNA. The results are in agreement with major groove contacts at positions two, three and five. At position four a low potential correlation of efficiencies with the H-bonding in the minor groove is found, while mutations at position six seem to influence repressor binding by other mechanisms.