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 previously against Drosophila melanogaster RNA polymerase II was mapped to amino 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 amino acid substitutions. The epitope is not involved in the polymerization reaction or the DNA unwinding process since no inhibitory effects of the monoclonal antibody were observed in nonspecific in vitro transcription using denatured calf thymus DNA or double stranded oligo dC-tailed T7 DNA as template. In contrast, MAb215 inhibits accurate in vitro transcription from the Krüppel 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 epitope does not participate directly in the formation of preinitiation complexes. The same inhibitory effects were observed using scFv215. The results provide further evidence that recombinant antibody fragments produced in Escherichia coli possess the same specificity and similar affinity as their parental antibodies and demonstrate that scFv fragments are useful tools for analysis of transcriptional processes.
Kontermann
R E
RE
Institute of Molecular Genetics, University of Heidelberg, Germany.
Liu
Z
Z
Schulze
R A
RA
Sommer
K A
KA
Queitsch
I
I
Dübel
S
S
Kipriyanov
S M
SM
Breitling
F
F
Bautz
E K
EK
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D003001:Cloning, Molecular; D004330:Drosophila / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D000939:Epitopes / Q000032:analysis*; D015151:Immunoblotting; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology; D008969:Molecular Sequence Data; D011485:Protein Binding; D012319:RNA Polymerase II / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012710:Serum Albumin, Bovine; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D012710:Serum Albumin, Bovine; D012319:RNA Polymerase II