Characterization of effects of anti-beta and anti-beta' monoclonal antibodies on the activity of the RNA polymerase from Escherichia coli.
Monoclonal antibodies directed against antigenic determinants on the beta and beta' subunits of the Escherichia coli RNA polymerase were characterized by using d(A-T)n-directed transcription assays. Antibodies were prepared by using purified subunits as immunogens, and seven anti-beta and five anti-beta' monoclonal antibodies were generated. Inhibitory anti-beta monoclonal antibodies were found to affect RNA polymerase during synthesis of r(A-U)n, abortive initiation of pApU and UpApU, and elongation by preformed ternary complexes. A comparative enzyme study of r(A-U)n synthesis showed the core polymerase to be more sensitive to inhibition by the anti-beta monoclonal antibody than was the holoenzyme. In contrast, the inhibition effected by the anti-beta' monoclonal antibody was found to be 90% or greater for each of the d(A-T)n-directed assays used. The different inhibitory patterns exhibited by the anti-beta and anti-beta' monoclonal antibodies suggest that the beta and beta' subunits engage in different roles during transcription. Kinetic analysis of the abortive initiation reaction in the presence and absence of the inhibitory antibodies resulted in distinctive but complex modes of inhibition. Inhibition by the anti-beta monoclonal antibody 210E8 was noncompetitive with regard to UTP and competitive for UpA incorporation; at increased UpA concentration, the inhibition was completely reversed. Inhibition of the abortive synthesis of UpApU by the anti-beta' monoclonal antibody 311G2 was noncompetitive with regard to both UpA and UTP incorporation. When the preformed ternary elongation complex was used, inhibition by the anti-beta monoclonal antibody was mixed with regard to the ribonucleoside triphosphate substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
D000911:Antibodies, Monoclonal; D012321:DNA-Directed RNA Polymerases / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D011063:Poly A-U / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D011063:Poly A-U; D012321:DNA-Directed RNA Polymerases