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 Rob's 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 Rob's CTD to SoxS and used indirect immunofluorescence microscopy to determine the effect of inducers on SoxS-Rob's 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, Rob's CTD serves as an anti-sigma factor in regulating the co-sigma-factor-like activity of SoxS when fused to it. Rob's CTD also protects its N-terminus from Lon protease, since Lon's normally rapid degradation of SoxS is blocked in the chimera. Accordingly, Rob's CTD has novel regulatory properties that can be bestowed on another E. coli protein.
Griffith
Kevin L
KL
University of Maryland Baltimore County, Baltimore, MD 21250, USA.
Fitzpatrick
M Megan
MM
Keen
Edward F
EF
Wolf
Richard E
RE
D004268:DNA-Binding Proteins / Q000378:metabolism*; D003652:Decanoic Acids / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D029968:Escherichia coli Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D049070:Protease La / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D015534:Trans-Activators / Q000032:analysis / Q000235:genetics; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D003652:Decanoic Acids; D029968:Escherichia coli Proteins; D011725:Pyridines; D011993:Recombinant Fusion Proteins; C080267:Rob protein, E coli; D015534:Trans-Activators; C070725:SoxS protein, E coli; C031071:decanoic acid; C483567:Lon protein, E coli; D049070:Protease La; C034306:4,4'-bipyridyl