EPITOPE MAPPING AND FUNCTIONAL-CHARACTERIZATION OF MONOCLONAL-ANTIBODIES SPECIFIC FOR THE ALPHA-SUBUNIT OF ESCHERICHIA-COLI RNA-POLYMERASE

The epitopes have been localized for a set of monoclonal antibodies specific for the alpha subunit of the Escherichia coli RNA polymerase. The antibodies are classified into three groups based on their epitopic assignments. Group 1, mAb 123C2, maps in the N terminus of cu between amino acids 1 and 23; Group 2 antibodies (mAb 129C4, mAb 124D1 and mAb 121C5) map in the central region between amino acids 190 and 210; Group 3 antibodies (mAb 130B1 and mAb 125C6) map in the C terminus between amino acids 310 and 320, mAb 130C2 is anomalous since it maps to the N terminus between amino acids 1 and 23 as well as to the C terminus between amino acids 320 and 329. The antibodies were used to investigate the role of cu in transcription activation with cAMP receptor protein dependent promoters. Three antibodies (130C2, 121C5, and 125C6) inhibited cAMP receptor protein-dependent initiation with lac P+ but not with lac UV5 or gal P+. Inhibition was observed with free RNA polymerase and the closed promoter complex; the preformed open promoter complex was insensitive. Only lac P+ was sensitive to these anti-alpha antibodies supporting the concept that the mode of interaction of RNA polymerase with cAMP receptor protein differs between lac P+ and gal PC.