Two Levels of Cooperativeness in the Binding of TodT to the tod Operon Promoter

• Published in: Journal of molecular biology 2008-12, Vol.384 (5), p.1037-1047
• Main Authors: Lacal, Jesús, Guazzaroni, María-Eugenia, Gutiérrez-del-Arroyo, Paloma, Busch, Andreas, Vélez, Marisela, Krell, Tino, Ramos, Juan L.
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding Sites; Calorimetry; Deoxyribonuclease I - metabolism; DNA Footprinting; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; Electrophoretic Mobility Shift Assay; Inverted Repeat Sequences - genetics; Microscopy, Atomic Force; Nucleic Acid Conformation; Operon - genetics; Peptide Fragments - metabolism; Promoter Regions, Genetic - genetics; Protein Binding; Pseudomonas; Pseudomonas putida; Pseudomonas putida - genetics; response regulator; sensor kinases; Thermodynamics; TodT; Trans-Activators - chemistry; Trans-Activators - metabolism; two-component systems
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2008.10.011

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.