Hierarchical Binding of the TodT Response Regulator to Its Multiple Recognition Sites at the tod Pathway Operon Promoter Jesús Lacal, María Eugenia Guazzaroni, Andreas Busch, Tino Krell and Juan L. Ramos⁎ Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda, 1, E-18008 Granada, Spain Received 29 August 2007; received in revised form 30 November 2007; accepted 4 December 2007 Available online 8 December 2007 The TodS and TodT proteins form a highly specific two-component regu- latory system that controls the expression of genes involved in the degradation of toluene, benzene, and ethylbenzene via the toluene dioxygenase pathway. The catabolic genes of the toluene dioxygenase pathway are transcribed from a single promoter called P todX once the response regulator TodT is phosphorylated by the TodS sensor kinase in response to pathway substrates. We show here that TodT is a monomer in solution and that it binds to three specific sites in the P todX promoter, centered at − 57, − 85, and − 106 with respect to the transcription start site. The − 85 and − 106 sites are pseudopalindromic, whereas the − 57 site is half a palindrome. TodT binding to its target sites is sequential, as shown by electrophoresis mobility gel shift assays and footprinting. The binding affinity values of TodT, as determined by isothermal titration calorimetry, are 1.8 ± 0.2, 5 ± 0.4, and 6.3 ± 0.8 μM for the − 106, − 85, and − 57 sites, respectively, and the binding stoichiometry is one monomer per half-palindromic element. Mutational analysis revealed that all three sites contribute to P todX strength, although the most relevant site is the distal one with respect to the − 10 extended element of the downstream promoter element. The C-TodT [C- terminal TodT fragment (amino acids 154–206)], a truncated variant of TodT that contains the C-terminal half of the protein bearing the DNA binding domain, binds in vitro to all three sites with affinity similar to that of the full- length protein. However, C-TodT, in contrast to the full-length regulator, does not activate in vitro transcription from P todX . We discuss the consequences of the organization of the binding sites on transcriptional control and propose that the N-terminal domain of TodT is necessary for appropriate interactions with other transcriptional elements. © 2007 Elsevier Ltd. All rights reserved. Edited by J. Karn Keywords: TodT; Pseudomonas; response regulator; sensor kinase; DNA– protein interactions Introduction Bacteria frequently respond to environmental cues by changing their pattern of transcription. 1 One of the systems bacteria most frequently use to sense chemical or physical changes in the environment is the two-component regulatory system (TCS), which typically consists of a histidine protein kinase that functions as an environmental sensor and a response regulator (RR) that mediates the transcriptional process. 2–6 The sensor kinase is usually autopho- sphorylated in response to cognate signals and sub- sequently transfers the phosphate to the RR protein. These regulators act as phosphorylation-activated switches that often mediate a cellular response through transcriptional regulation. 7,8 Among the processes regulated by TCS in bacteria are host *Corresponding author. E-mail address: jlramos@eez.csic.es. Abbreviations used: C-TodT, C-terminal TodT fragment (amino acids 154–206); EMSA, electrophoretic mobility shift assay; HTH, helix–turn–helix; IHF, integration host factor; ITC, isothermal titration calorimetry; RR, response regulator; TCS, two-component regulatory system. Available online at www.sciencedirect.com doi:10.1016/j.jmb.2007.12.004 J. Mol. Biol. (2008) 376, 325–337 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.