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.
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 ion-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.
Strickland
M S
MS
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Thompson
N E
NE
Burgess
R R
RR
D000911:Antibodies, Monoclonal / Q000276:immunology*; D012321:DNA-Directed RNA Polymerases / Q000276:immunology*; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology; D010446:Peptide Fragments / Q000276:immunology; D010449:Peptide Mapping; D012808:Sigma Factor / Q000276:immunology*; D014157:Transcription Factors / Q000276:immunology*; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D012808:Sigma Factor; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases