Inhibitory monoclonal antibody to calf thymus RNA polymerase II blocks formation of enzyme-DNA complexes.
Monoclonal antibodies were prepared against purified calf thymus RNA polymerase II (RNA nucleotidyltransferase, EC 2.7.7.6). Two distinct antibodies were identified on the basis of their ability to bind RNA polymerase II in a solid-phase assay and to remove enzyme activity from solution in complexes precipitated by anti-immunoglobulin antibodies. One monoclonal antibody directly inhibited enzyme activity in solution. Detailed study of the mechanism of enzyme inhibition by this antibody revealed that it prevented the formation of enzyme-DNA complexes and had no effect on chain elongation. Inhibition of DNA binding and catalytic activity depended on preincubation of antibody with enzyme and was irreversible. Without preincubation, the enzyme readily bound to DNA in the presence of antibody, indicating that the binding of the free enzyme to DNA is faster than that of the antibody to the enzyme. In a competitive immunoassay enzyme-DNA complexes were much less reactive with antibody than was free enzyme, suggesting that DNA either blocks the antigenic site or alters the enzyme's conformation, preventing antibody recognition. These antibodies may serve as useful reagents for analysis of RNA polymerase structure and function.
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002417:Cattle; D004247:DNA / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000276:immunology*; D000939:Epitopes; D011485:Protein Binding; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors / Q000276:immunology*; D013329:Structure-Activity Relationship; D013950:Thymus Gland / Q000201:enzymology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D004247:DNA; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases