Two levels of cooperativeness in the binding of TodT to the tod operon promoter.

The TodS/TodT two-component system controls the expression of tod genes for toluene degradation in Pseudomonas putida. TodT binds to two pseudopalindromes at -106 (Box-1) and -85 (Box-2), as well as to a half-palindrome (Box-3), with respect to the main transcription initiation site in the PtodX promoter. TodT recognizes each half-palindrome in Boxes-1 and -2, but affinities for these sequences are lower than those for the pseudopalindromes, pointing towards positive cooperativeness in intrabox recognition. TodT's affinity for DNA fragments containing two vicinal boxes (either Boxes-1 and -2 or Boxes-2 and -3) is higher than its affinity for individual boxes, suggesting interbox cooperativeness. Similar patterns of cooperativeness were observed for the recombinant TodT DNA-binding domain [C-terminal TodT fragment (aa 154-206) (C-TodT)], suggesting important cooperativeness determinants in this domain. Occupation of PtodX by TodT is initiated at Box-1, and optimization of its palindromic order increases affinity in vitro; however, this does not result in enhanced in vivo gene expression. Mutations at either half of the Box-1 palindrome have no significant effects on transcriptional activity, whereas mutations in the entire Box-1 cause a 12-fold reduction. Using atomic force microscopy, we show that TodT induces a DNA hairpin bend at PtodX between Boxes-2 and -3, as supported by footprint studies showing a hyperreactive nucleotide at G -68. The N-terminal part of TodT seems to play a central role in hairpin formation, since C-TodT neither induces a bend nor causes G -68 hyperreactivity in footprints. This hairpin seems important for transcriptional activation, since C-TodT binding to PtodX does not stimulate transcription.