title: The anti-sigma factor SpoIIAB forms a 2:1 complex with sigma(F), contacting multiple conserved regions of the sigma factor.
abstract: The developmental regulatory protein sigma(F) of Bacillus subtilis, a member of the sigma(70)-family of bacterial RNA polymerase sigma factors, is negatively regulated by the anti-sigma factor SpoIIAB, which binds to sigma(F), sequestering it in an inactive complex. SpoIIAB binding to sigma(F) is strongly stimulated by ATP. Here, we use a combination of gel filtration chromatography, dynamic light-scattering, analytical ultracentrifugation, limited proteolysis with N-terminal sequencing and electrospray mass spectrometry, and deletion analysis to probe the SpoIIAB-sigma(F) complex. The studies were facilitated by investigating the homologs from Bacillus stearothermophilus as well as co-expression of the proteins in Escherichia coli, allowing purification of large quantities of the in vivo assembled complex. We determined the stoichiometry of the complex to be SpoIIAB(2):sigma(F)(1). Alone, sigma(F) is rapidly degraded by the protease trypsin. In the complex with SpoIIAB, however, sigma(F) is remarkably resistant to proteolysis. Analysis of the protease cleavage data indicates the anti-sigma binds sigma(F) through contacts with mutliple conserved regions of the sigma factor, supporting previous findings based on genetic data.
authors.affiliation: The Rockefeller University, 1230 York Avenue, New York, NY, 10021, USA.
authors.forename: E A
authors.initials: EA
authors.lastname: Campbell
authors.forename: S A
authors.initials: SA
authors.lastname: Darst
mesh_terms: D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002850:Chromatography, Gel; D017124:Conserved Sequence / Q000235:genetics; D001411:Geobacillus stearothermophilus; D008027:Light; D013058:Mass Spectrometry; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D017384:Sequence Deletion / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014157:Transcription Factors; D014357:Trypsin / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus; D014357:Trypsin