Roles of effectors in XylS-dependent transcription activation: intramolecular domain derepression and DNA binding.
XylS, an AraC family protein, activates transcription from the benzoate degradation pathway Pm promoter in the presence of a substrate effector such as 3-methylbenzoate (3MB). We developed a procedure to obtain XylS-enriched preparations which proved suitable to analyze its activation mechanism. XylS showed specific 3MB-independent binding to its target operator, which became strictly 3MB dependent in a dimerization-defective mutant. We demonstrated that the N-terminal domain of the protein can make linker-independent interactions with the C-terminal domain and inhibit its capacity to bind DNA. Interactions are hampered in the presence of 3MB effector. We propose two independent roles for 3MB in XylS activation: in addition to its known influence favoring protein dimerization, the effector is able to modify XylS conformation to trigger N-terminal domain intramolecular derepression. We also show that activation by XylS involves RNA polymerase recruitment to the Pm promoter as demonstrated by chromatin immunoprecipitation assays. RNA polymerase switching in Pm transcription was reproduced in in vitro transcription assays. All sigma(32)-, sigma(38)-, and sigma(70)-dependent RNA polymerases were able to carry out Pm transcription in a rigorous XylS-dependent manner, as demonstrated by the formation of open complexes only in the presence of the regulator.
Domínguez-Cuevas
Patricia
P
Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Department of Environmental Protection, Granada, Spain.
Marín
Patricia
P
Busby
Stephen
S
Ramos
Juan L
JL
Marqués
Silvia
S
D001426:Bacterial Proteins / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001565:Benzoates / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary; D012091:Repetitive Sequences, Nucleic Acid; D015534:Trans-Activators / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D001565:Benzoates; D004268:DNA-Binding Proteins; D015534:Trans-Activators; C078720:XylS protein, Pseudomonas putida; C044605:methyl benzoate; D004247:DNA; D012321:DNA-Directed RNA Polymerases