CHARACTERIZATION OF THE EPITOPE RECOGNIZED BY A MONOCLONAL-ANTIBODY DIRECTED AGAINST THE LARGEST SUBUNIT OF DROSOPHILA RNA-POLYMERASE-II

The epitope recognized by monoclonal antibody MAb215 generated previou sly against Drosophila melanogaster RNA polymerase II was mapped to am ino acid residues 806-820 of the largest, 215 kDa, subunit located in a region conserved within the largest subunits of pro- and eukaryotic RNA polymerases, The affinities of MAb215 and of a recombinant single- chain Fv fragment (scFv215) were determined for binding to the enzyme as well as the fusion protein and synthetic peptides used for epitope mapping, In addition, amino acid residues of the epitope important for binding to MAb215 were identified using peptides carrying single amin o acid substitutions, The epitope is not involved in the polymerizatio n reaction or the DNA unwinding process since no inhibitory effects of the monoclonal antibody were observed in nonspecific in vitro transcr iption using denatured calf thymus DNA or double stranded oligo dC-tai led T7 DNA as template, In contrast, MAb215 inhibits accurate in vitro transcription from the Kruppel gene promoter and from the adenovirus- 2 major late promoter, Preincubation of template DNA with the nuclear extract had no effects on inhibition supporting the notion that the ep itope does not participate directly in the formation of preinitiation complexes, The same inhibitory effects were observed using scFv215, Th e results provide further evidence that recombinant antibody fragments produced in Escherichia coil possess the same specificity and similar affinity as their parental antibodies and demonstrate that scFv fragm ents are useful tools for analysis of transcriptional processes.