Characterization of MobR, the 3-Hydroxybenzoate-responsive Transcriptional Regulator for the 3-Hydroxybenzoate Hydroxylase Gene of Comamonas testosteroni KH122-3s

Autor: Keiichi Hosokawa, Hanako Matsue, Hiroshi Yamaguchi, Mariko Yoshida, Hiroki Higashibata, Takeshi Tanaka, Takeshi Hiromoto, Shinsuke Fujiwara
Rok vydání: 2006
Předmět:
Operator Regions
Genetic
Transcription
Genetic
Protein Conformation
Molecular Sequence Data
DNA Footprinting
Parabens
Repressor
Electrophoretic Mobility Shift Assay
Polymerase Chain Reaction
Primer extension
Mixed Function Oxygenases
Allosteric Regulation
Bacterial Proteins
Start codon
Structural Biology
Escherichia coli
Hydroxybenzoates
Amino Acid Sequence
Comamonas testosteroni
Cloning
Molecular
Promoter Regions
Genetic
Molecular Biology
Binding Sites
Base Sequence
Sequence Homology
Amino Acid
biology
Circular Dichroism
Promoter
Gene Expression Regulation
Bacterial
4-Hydroxybenzoate-3-Monooxygenase
biology.organism_classification
Recombinant Proteins
Open reading frame
Biochemistry
Hydroxybenzoate
Trans-Activators
Transcription Initiation Site
Salicylic Acid
Energy source
Dimerization
Plasmids
Zdroj: Journal of Molecular Biology. 364:863-877
ISSN: 0022-2836
DOI: 10.1016/j.jmb.2006.08.098
Popis: Comamonas testosteroni KH122-3s is an aerobic soil bacterium that utilizes 3-hydroxybenzoate as a sole carbon and energy source. In this strain, 3-hydroxybenzoate hydroxylase (MobA) acts on the initial step of the degradation to produce 3,4-dihydroxybenzoate, which is subsequently subjected to the meta-cleavage pathway leading to tricarboxylic acid cycle intermediates. Gene walking analysis of the upstream region of mobA revealed an open reading frame (mobR) that encodes a transcriptional regulator of the MarR family. Here, we report that MobR negatively regulates the expression of mobA, and that the repression is relieved by binding of 3-hydroxybenzoate, the substrate for MobA. A primer extension experiment was performed to determine the transcription start site for mobA and identified it at 83 bp upstream of the mobA start codon, accompanied by a typical sigma70-type promoter. The mobR gene was expressed in Escherichia coli cells and the recombinant product was purified to homogeneity. Gel mobility-shift assays and DNase I footprinting analyses indicated that MobR binds as a homodimer to an imperfect inverted repeat within the mobA-mobR intergenic region, with an apparent dissociation constant of 11.5(+/- 0.5) nM. The operator site is located between the start codon and the promoter region for mobA, suggesting that MobR functions as a transcriptional repressor for mobA expression. The results of effector-binding assays indicated that MobR, but not its isomers 4-hydroxybenzoate and salicylate, is released from the operator site by the addition of 3-hydroxybenzoate. This dissociation process is highly cooperative, with a Hill coefficient of approximately 2. In addition, CD spectroscopic studies demonstrated that MobR adopts two conformational states corresponding to the effector-bound and unbound forms. These results suggest that the MobR dimer possesses at least two effector-binding sites, and that the effector binding to MobR induces an allosteric conformational change required for dissociation of the protein-DNA complex.
Databáze: OpenAIRE
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