Signaling properties of the sigma E activation pathway

sigmaE activity is required to diminish general stress in the envelope of Escherichia coli and to ensure proper insertion of outer membrane porins (OMP) into the outer membrane. Accumulation of unassembled OMP monomers in the periplasm generates a signal that is communicated through the inner membrane to the cytoplasm, where it activates the transcription factor, sigmaE. The signal is conveyed by proteolytic cascade that carries out regulated proteolysis of RseA, a membrane-spanning anti-sigma factor whose cytoplasmic domain inhibits sigmaE-dependent transcription. Upon binding to OMP C-termini, the membrane localized DegS protease is activated to cleave RseA in its periplasmic domain. The membrane embedded protease, RseP, then cleaves RseA close to the inner membrane, and sigma E is released in complex with cytoplasmic RseA fragment that is subsequently degraded. The transcriptional activity of sigmaE varies over 40-fold in a graded manner depending on the expression level of OMP. In this thesis I have addressed the organization of the pathway. First, I have shown that sigmaE activity correlates with the rate of RseA proteolysis because RseA degradation determines the rate of sigmaE release into the cytoplasm. This mechanism enables cells to respond to the changes in RseA degradation rate rapidly, prior to changes in the relative levels of RseA and sigmaE. I also present evidence that RseB (a periplasmic protein) and DegS function to make the system sensitive to a wide range of OMP concentrations and unresponsive to variations in the levels of DegS and RseP proteases. These features rely on the inability of RseP to cleave intact RseA. I demonstrate that RseB, which binds to the periplasmic face of RseA, and DegS each independently inhibit RseP cleavage of intact RseA.; In a separate project, I measured the levels of RNA polymerase, sigma 70, sigmaE and sigma32 in E. coli. I use these values in an equilibrium model that considers RNA polymerase binding to DNA to explore theoretically two aspects of general regulation of transcription: recruitment of RNA polymerase to promoters by activators and competition between sigma's.