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Characterization of monoclonal antibodies against Escherichia coli core RNA polymerase
Jöelle Rouby1, Martine Pugniere, Jean-Claude Mani
1Centre National de la Recherche Scietifique (CNRS), Unité Mixte de Recherche (UMR) 5094, Institut de Biotechnologie et Pharmacologie, Faculté de Pharmacie, 15 Avenue Charles Flahault, 34060 Montpellier Cedex 2, France.
Abstract:
Multiple interactions with DNA, RNA and transcription factors occur in a transcription cycle. To survey the proximity of some of these factors to the Escherichia coli RNA polymerase surface, we produced a set of nine monoclonal antibodies (mAbs) against the enzyme. These mAbs, located at different places on the surface of the enzyme, were used in a co-immunopurification assay to investigate interference with the binding of NusA, sigma70, GreB and HepA to core RNA polymerase. One of these mAbs turned out to be the first antibody inhibitor of the binding of NusA and sigma70; it did not affect GreB and HepA interactions. Its epitope was located on the beta' subunit at the C-terminus of region G. The properties of this mAb reinforce the idea that the mutually exclusive binding of NusA and sigma70 to core RNA polymerase is due to, at least partially, overlapping binding sites, rather than allosteric interaction between two distant binding sites. This mAb is also useful to understand the occupancy of sigma70, NusA and Gre proteins on core RNA polymerase.
Insights
Researchers developed monoclonal antibodies (mAbs) to study Escherichia coli RNA polymerase interactions. One mAb inhibits NusA and sigma70 binding, supporting overlapping binding sites on the RNA polymerase beta' subunit.
Area of Science:
- Molecular Biology
- Biochemistry
- Microbiology
Background:
- Transcription involves complex interactions between DNA, RNA, and transcription factors with RNA polymerase.
- Understanding the spatial proximity of these factors to RNA polymerase is crucial for elucidating transcription regulation.
Purpose of the Study:
- To investigate the binding interactions of transcription factors NusA, sigma70, GreB, and HepA with Escherichia coli core RNA polymerase.
- To identify specific epitopes on RNA polymerase that mediate or interfere with these interactions.
Main Methods:
- Production of nine unique monoclonal antibodies (mAbs) targeting the surface of Escherichia coli RNA polymerase.
- Co-immunopurification assays to assess the impact of mAbs on the binding of transcription factors to core RNA polymerase.
Main Results:
- One mAb was identified as the first antibody inhibitor of NusA and sigma70 binding to core RNA polymerase.
- This inhibitory mAb did not affect GreB or HepA binding, indicating specific interference.
- The epitope for the inhibitory mAb was mapped to the C-terminus of region G on the beta' subunit of RNA polymerase.
Conclusions:
- The findings support the model of mutually exclusive binding between NusA and sigma70, likely due to overlapping binding sites on the RNA polymerase beta' subunit.
- This specific mAb serves as a valuable tool for studying the occupancy and interactions of sigma70, NusA, and Gre proteins on core RNA polymerase.