Campbell E A, Darst S A
The Rockefeller University, 1230 York Avenue, New York, NY, 10021, USA.
J Mol Biol. 2000 Jun 30;300(1):17-28. doi: 10.1006/jmbi.2000.3838.
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.
