Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1982 Dec;79(23):7233–7237. doi: 10.1073/pnas.79.23.7233

Inhibitory monoclonal antibody to calf thymus RNA polymerase II blocks formation of enzyme-DNA complexes.

S B Carroll, B D Stollar
PMCID: PMC347313  PMID: 6185948

Abstract

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.

Full text

PDF
7233

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrzejewski C., Jr, Stollar B. D., Lalor T. M., Schwartz R. S. Hybridoma autoantibodies to DNA. J Immunol. 1980 Mar;124(3):1499–1502. [PubMed] [Google Scholar]
  2. Brodner O. G., Wieland T. Identification of the amatoxin-binding subunit of RNA polymerase B by affinity labeling experiments. Subunit B3-the true amatoxin receptor protein of multiple RNA polymerase B. Biochemistry. 1976 Aug 10;15(16):3480–3484. doi: 10.1021/bi00661a013. [DOI] [PubMed] [Google Scholar]
  3. Buhler J. M., Huet J., Davies K. E., Sentenac A., Fromageot P. Immunological studies of yeast nuclear RNA polymerases at the subunit level. J Biol Chem. 1980 Oct 25;255(20):9949–9954. [PubMed] [Google Scholar]
  4. Christmann J. L., Dahmus M. E. Monoclonal antibody specific for calf thymus RNA polymerases IIO and IIA. J Biol Chem. 1981 Nov 25;256(22):11798–11803. [PubMed] [Google Scholar]
  5. Greenleaf A. L., Weeks J. R., Voelker R. A., Ohnishi S., Dickson B. Genetic and biochemical characterization of mutants at an RNA polymerase II locus in D. melanogaster. Cell. 1980 Oct;21(3):785–792. doi: 10.1016/0092-8674(80)90441-9. [DOI] [PubMed] [Google Scholar]
  6. Hinkle D. C., Chamberlin M. J. Studies of the binding of Escherichia coli RNA polymerase to DNA. I. The role of sigma subunit in site selection. J Mol Biol. 1972 Sep 28;70(2):157–185. doi: 10.1016/0022-2836(72)90531-1. [DOI] [PubMed] [Google Scholar]
  7. Hinkle D. C., Chamberlin M. J. Studies of the binding of Escherichia coli RNA polymerase to DNA. II. The kinetics of the binding reaction. J Mol Biol. 1972 Sep 28;70(2):187–195. doi: 10.1016/0022-2836(72)90532-3. [DOI] [PubMed] [Google Scholar]
  8. Hodo H. G., 3rd, Blatti S. P. Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II. Biochemistry. 1977 May 31;16(11):2334–2343. doi: 10.1021/bi00630a005. [DOI] [PubMed] [Google Scholar]
  9. Huet J., Sentenac A., Fromageot P. Spot-immunodetection of conserved determinants in eukaryotic RNA polymerases. Study with antibodies to yeast RNA polymerases subunits. J Biol Chem. 1982 Mar 10;257(5):2613–2618. [PubMed] [Google Scholar]
  10. Ingles C. J. Antigenic homology of eukaryotic RNA polymerases. Biochem Biophys Res Commun. 1973 Nov 16;55(2):364–371. doi: 10.1016/0006-291x(73)91096-6. [DOI] [PubMed] [Google Scholar]
  11. Krämer A., Bautz E. K. Immunological relatedness of subunits of RNA polymerase II from insects and mammals. Eur J Biochem. 1981 Jul;117(3):449–455. doi: 10.1111/j.1432-1033.1981.tb06359.x. [DOI] [PubMed] [Google Scholar]
  12. Krämer A., Haars R., Kabisch R., Will H., Bautz F. A., Bautz E. K. Monoclonal antibody directed against RNA polymerase II of Drosophila melanogaster. Mol Gen Genet. 1980;180(1):193–199. doi: 10.1007/BF00267369. [DOI] [PubMed] [Google Scholar]
  13. Ruet A., Sentenac A., Fromageot P., Winsor B., Lacroute F. A mutation of the B220 subunit gene affects the structural and functional properties of yeast RNA polymerase B in vitro. J Biol Chem. 1980 Jul 10;255(13):6450–6455. [PubMed] [Google Scholar]
  14. Weeks J. R., Coulter D. E., Greenleaf A. L. Immunological studies of RNA polymerase II using antibodies to subunits of Drosophila and wheat germ enzyme. J Biol Chem. 1982 May 25;257(10):5884–5892. [PubMed] [Google Scholar]
  15. Weil P. A., Blatti S. P. Partial purification and properties of calf thymus deoxyribonucleic acid dependent RNA polymerase III. Biochemistry. 1975 Apr 22;14(8):1636–1642. doi: 10.1021/bi00679a015. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES