Hierarchical binding of the TodT response regulator to its multiple recognition sites at the tod pathway operon promoter.
The TodS and TodT proteins form a highly specific two-component regulatory 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 microM 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.
Lacal
Jesús
J
Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda, 1, E-18008 Granada, Spain.
Guazzaroni
María Eugenia
ME
Busch
Andreas
A
Krell
Tino
T
Ramos
Juan L
JL
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009876:Operon; D010105:Oxygenases / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C104650:TodT protein, Pseudomonas putida; D015534:Trans-Activators; D010105:Oxygenases; C023172:toluene dioxygenase; D001616:beta-Galactosidase