Immunological studies of yeast nuclear RNA polymerases at the subunit level

J M Buhler, J Huet, K E Davies, A Sentenac, P Fromageot
1980 Journal of Biological Chemistry  
Antisera were raised against native RNA polymerases A or B, as well as against each individual subunit of RNA polymerase A from the yeast Saccharmoyces cerevisiae. The affinity spectrum of antibodies was evaluated by reacting electrophoretically separated enzyme subunits, transferred to a membrane, with 125I-labeled immunoglobulins. Alternatively, the subunit . immunoglobulin complex was revealed by 125I-labeled Protein A. Antibodies directed against native RNA polymerase A recognized the
more » ... ecognized the majority of the polypeptides forming the enzyme. When challenged with RNA polymerases B or C, this antibody preparation demonstrated the presence of polypeptides common to the three enzymes. A small cross-reaction was also found at the level of the large subunits of Enzyme B as well as some additional polypeptides of Enzyme C. Similar experiments with antibodies directed against native RNA polymerase B confirmed the presence of common subunits and also showed that the large polypeptides of the three enzymes share a few immunological determinants. Common subunits are AC40, ABC27, ABC23, AC19, and ABC14.5. Immunologically related sites were conserved in the large subunits of RNA polymerase A from remote yeast species. Similarly, yeast and wheat germ RNA polymerase B share immunological determinants on the large subunit as well as on a small peptide. On the other hand, there was no significant cross-reaction between yeast and mammalian Enzyme B or Escherichia coli RNA polymerase. Antibodies raised against the different polypeptide components of RNA polymerase A reacted specifically with the corresponding subunits. Inhibition studies with these subunit-specific antibodies showed that the common subunits are not always similarly exposed to antibody attack within the three enzymes. The data are discussed in terms of the structural similarity, organization and evolution of eukaryotic RNA polymerases.
pmid:7000767 fatcat:6yhqb5vu3bgsxibzcpd3qlvvpy