Characterization and epitope mapping of monoclonal antibodies directed against the beta' subunit of the Escherichia coli RNA polymerase.
Monoclonal antibodies (mAbs) raised against the beta' subunit of the Escherichia coli RNA polymerase were used to probe the structure and function of this subunit. Of the five anti-beta' monoclonal antibodies studied, only mAb 311G2 is a strong inhibitor of RNA polymerase activity. This antibody binds to an epitope which is exposed in both the assembled holoenzyme and isolated beta' subunit. In contrast, the null antibodies bind to the free beta' subunit but very weakly to native RNA polymerase. It would appear that the beta' domain in which their epitopes reside is either conformationally altered or blocked due to interaction with other subunits in native RNA polymerase. In order to locate the positions of the epitopes for these five monoclonal antibodies, a series of overlapping deletion mutants have been constructed by partial restriction and religation of the beta' gene present in pT7 beta' (Zalenskaya, K., Lee, J., Gujuluva, C. N., Shin, Y. K., Slutsky, M., nd Goldfarb, A. (1990) Gene 89, 7-12). The presence of the epitopes for each of the anti-beta' monoclonal antibodies was assessed by Western blotting. The results indicate that the epitopes for mAb 340F11, mAb 370F3, mAb 371D6, and mAb 372B2 are located between amino acids 817-876. This region may be important in enzyme assembly or subunit-subunit interaction. The epitope for the inhibitory antibody, mAb 311G2, is located between amino acids 1047-1093. This region may be involved in the catalytic function of RNA polymerase.
Luo
J
J
Department of Biological Sciences, Hunter College of the City University, New York, New York 10021.
Krakow
J S
JS
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D015153:Blotting, Western; D002872:Chromosome Deletion; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D015183:Restriction Mapping
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D012321:DNA-Directed RNA Polymerases