Dominant negative mutations in the Tn10 tet repressor: evidence for use of the conserved helix-turn-helix motif in DNA binding

Abstract

The Tn10 tet repressor regulates transcription of the tetracycline-resistance determinant in transposon Tn10. Previous DNA sequencing studies identified a region of tet repressor (amino acids 26-47) that is homologous to the helix-turn-helix regions of lambda Cro, lambda repressor, and catabolite gene activator protein that are implicated in sequence-specific DNA binding. Here we report the isolation of dominant tetR mutations that result in tet repressors deficient in tet operator binding but that retain some capacity to form dimers with, and thereby inactivate, wild-type repressor monomers. The mutations were isolated by transforming a tetR+ tetA-lacZ fusion strain with hydroxylamine-mutagenized tetR plasmid DNA and then screening for increased lacZ expression. DNA sequence analysis of 35 independent isolates identified seven different mutations, five of which are in the region of helix-turn-helix sequence homology. In vitro binding studies indicate that the mutations in this region of tet repressor reduce the affinity of tet repressor for tet operator DNA by at least a factor of 1000 but have no significant effect on the affinity of tet repressor for tetracycline. These results provide strong support for the proposal that tet repressor utilizes the conserved helix-turn-helix structural motif in binding to tet operator DNA.