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 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.

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth

“…While the phenotypes caused by the csh203 insertion mutation are almost certainly due to a decrease in the intracellular concentration of sigma-A, we could detect little or no difference between the amounts of sigma-A in the csh203 mutant and the wild type with a monoclonal antibody raised against sigma-70 of E. coli (provided by R. Burgess [30]). A twofold change in the amount of sigma-A would have been detected (data not shown).…”

The Alternative Sigma Factor σBofBacillus cereus: Response to Stress and Role in Heat Adaptation

“…Even when the histidine tag was taken into account, B migrated as a slightly larger protein in the SDS-PAGE gel than predicted on the basis of its predicted molecular mass (29 kDa). This is a property of many sigma factors because of highly positive and negative charge clusters in the proteins (6,13,15,42). The antiserum that was raised against B reacted specifically with purified B , although some crossreaction was also seen with larger proteins in the purified B sample (Fig.…”

Alternative transcription factor sigmaSB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar

“…Both the sigB genes of B. subtilis and S. aureus encode proteins with a calculated molecular mass of Ϸ30 kDa (7,16,40). The factors usually migrate abnormally on SDS-polyacrylamide gels because of highly positive and negative charge clusters (18,35). SigB of B. subtilis migrates as a protein of 37 kDa on an SDS-polyacrylamide gel (21).…”