Two Functions of the C-Terminal Domain of Escherichia coli Rob: Mediating “Sequestration–Dispersal” as a Novel Off–On Switch for Regulating Rob’s Activity as a Transcription Activator and Preventing Degradation of Rob by Lon Protease

In Escherichia coli, Rob activates transcription of the SoxRS/MarA/Rob regulon. Previous work revealed that Rob resides in three to four immunostainable foci, that dipyridyl and bile salts are inducers of its activity, and that inducers bind to Robʹs C-terminal domain (CTD). We propose that sequestration inactivates Rob by blocking its access to the transcriptional machinery and that inducers activate Rob by mediating its dispersal, allowing interaction with RNA polymerase. To test “sequestration–dispersal” as a new mechanism for regulating the activity of transcriptional activators, we fused Robʹs CTD to SoxS and used indirect immunofluorescence microscopy to determine the effect of inducers on SoxS–Robʹs cellular localization. Unlike native SoxS, which is uniformly distributed throughout the cell, SoxS–Rob is sequestered without an inducer, but is rapidly dispersed when cells are treated with an inducer. In this manner, Robʹs CTD serves as an anti-sigma factor in regulating the co-sigma-factor-like activity of SoxS when fused to it. Robʹs CTD also protects its N-terminus from Lon protease, since Lonʹs normally rapid degradation of SoxS is blocked in the chimera. Accordingly, Robʹs CTD has novel regulatory properties that can be bestowed on another E. coli protein.