Structure and function of the .sigma.-70 subunit of Escherichia coli RNA polymerase. Monoclonal antibodies: localization of epitopes by peptide mapping and effects on transcription

Abstract

Murine monoclonal antibodies reactive with the major .sigma. subunit (.sigma.-70) of Escherichia coli RNA polymerase were obtained by standard hybridoma techniques. Western blot analyses established that seven antibodies had unique specificities after various chemical and enzymatic methods were used to fragment .sigma.. Peptides were purified by HPLC using size-exclusion, reverse-phase, or iron-exchange chromatography. The epitopes for six of these antibodies have been localized to specific peptides. These peptides were further characterized by amino acid composition and N-terminal sequencing. .sigma., which has a molecular weight of 70.2K, runs as 83K on SDS gels in this study. This anomalous behavior has been localized to the very acidic N-terminal half of the molecule. One antibody is unable to bind to native .sigma.. Two others do not bind well to .sigma. when it is contained in holoenzyme, indicating that their epitopes are in regions of .sigma. which are inaccessible in the holoenzyme complex. All three of these antibodies fail to inhibit in vitro transcription by holoenzyme. The other four antibodies all can inhibit in vitro transcription.