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TOL plasmid transcription factor XylS binds specifically to the Pm operator sequence
Molecular Microbiology
|May 1, 1996
Summary
XylS protein specifically binds to the Pseudomonas putida TOL plasmid meta operon promoter (Pm) operator (Om). This binding, involving two DNA-binding units, is crucial for activating gene transcription.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- XylS, an AraC family transcription factor, regulates the Pseudomonas putida TOL plasmid meta operon via the Pm promoter.
- A specific DNA sequence (tandem homologous direct repeats) is essential for XylS-mediated Pm activation in vivo.
Purpose of the Study:
- To characterize the specific binding of XylS to the Pm operator (Om).
- To elucidate the molecular mechanism of XylS-DNA interaction.
Main Methods:
- Overexpression and immunopurification of epitope-tagged XylS (N-XylS).
- DNA footprinting assays (DNase I and hydroxyl radical).
- Methylation interference experiments.
Main Results:
- N-XylS specifically binds to the Om region.
- XylS binding protects a 44 bp region on both DNA strands.
- XylS contacts DNA along one side, covering four helical turns with base-specific interactions in major groove regions.
- Binding site arrangement and DNA contact pattern resemble those of the E. coli AraC protein.
Conclusions:
- XylS binds to the Pm operator in a manner consistent with two DNA-binding units.
- The findings provide insights into the transcriptional regulation mechanism of the TOL plasmid meta operon by XylS.