The quorum‐sensing transcription factor TraR decodes its DNA binding site by direct contacts with DNA bases and by detection of DNA flexibility

Abstract

TraR of Agrobacterium tumefaciens is a member of the LuxR family of transcriptional regulators, and binds to specific DNA sequences (tra boxes) at target promoters of the tumour-inducing (Ti) plasmid. Each tra box has a pronounced dyad symmetry, and each subunit of a TraR dimer binds to one half of a tra box via a helix-turn-helix (HTH) DNA binding motif. Structural analysis has suggested that TraR makes extensive sequence-specific contacts with tra box DNA. In this study, we tested these predictions using synthetic self-complementary oligonucleotides containing variant tra box sequences. Some predictions made from structural analysis were confirmed, while others were shown to be incorrect. Unexpectedly, these experiments also showed that six nucleotides at the centre of the tra box that make no direct contact with TraR are nevertheless critical for high-affinity binding and probably act by facilitating a previously described DNA bend. Variant tra boxes were also tested for transcription activity in vivo. Most transcription assays reflected in vitro binding assays. However, alterations of the outermost nucleotides had little effect on TraR binding but blocked transcription, probably by altering an overlapping -35 promoter motif.