Physical evidence for the induced release of the Bacillus subtilis transcription factor, sigma(F), from its inhibitory complex.
The release of the transcription factor sigma(F) from its inhibitory complex with SpoIIAB is a key regulatory step in the control of sporulation in Bacillus subtilis as it initiates a pattern of differential gene expression in the mother cell and prespore compartments. The sigma(F).SpoIIAB complex is dissociated by the unphosphorylated form of the protein SpoIIAA, the alternative binding partner of SpoIIAB. Here, we employ fluorescence spectroscopy to examine the mechanism by which SpoIIAA acts on the sigma(F).SpoIIAB complex. We constructed a mutant of sigma(F), sigma(F)-W46L, which displayed a reproducible fluorescence response on binding to SpoIIAB. Using this mutant we were able to quantify the amount of sigma(F) bound to SpoIIAB in real time. The results provide physical evidence for the "induced release" mechanism of sigma(F) activation. We demonstrate that SpoIIAA interacts directly with the sigma(F).SpoIIAB complex, greatly decreasing the affinity of SpoIIAB for sigma(F) and thus causing the release of the latter. We also demonstrate that sigma(F) is released before SpoIIAA is phosphorylated and that release occurs on a similar time scale to the binding of SpoIIAA to SpoIIAB.
Clarkson
Joanna
J
Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Campbell
Iain D
ID
Yudkin
Michael D
MD
D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D007700:Kinetics; D015203:Reproducibility of Results; D012808:Sigma Factor / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D017931:DNA Primers; C064971:FliA protein, Bacteria; D012808:Sigma Factor; C030069:spore-specific proteins, Bacillus