LOCALIZATION OF YEAST RNA-POLYMERASE-I CORE SUBUNITS BY IMMUNOELECTRON MICROSCOPY

Immunoelectron microscopy was used to determine the spatial organizati on of the yeast RNA polymerase I core subunits on a three-dimensional model of the enzyme. Images of antibody-labeled enzymes were compared with the native enzyme to determine the localization of the antibody b inding site on the surface of the model. Monoclonal antibodies were us ed as probes to identify the two largest subunits homologous to the ba cterial beta and beta' subunits. The epitopes for the two monoclonal a ntibodies were mapped using subunit-specific phage display libraries, thus allowing a direct correlation of the structural data with functio nal information on conserved sequence elements. An epitope close to co nserved region C of the beta-like subunit is located at the base of th e finger-like domain, whereas a sequence between conserved regions C a nd D of the beta'-like subunit is located in the apical region of the enzyme. Polyclonal antibodies outlined the alpha-like subunit AC(40) a nd subunit AC(19) which were found colocalized also in the apical regi on of the enzyme. The spatial location of the subunits is correlated w ith their biological activity and the inhibitory effect of the antibod ies.