Characterization of monoclonal antibodies against Escherichia coli core RNA polymerase.
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.
Rouby
Jöelle
J
Centre 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.
Pugniere
Martine
M
Mani
Jean-Claude
JC
Granier
Claude
C
Monmouton
Pierrette
P
Theulier Saint Germain
Stephane
S
Leonetti
Jean-Paul
JP
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D001483:Base Sequence; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D004926:Escherichia coli / Q000201:enzymology*; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases