Roles of glucitol in the GutR-mediated transcription activation process in Bacillus subtilis: tight binding of GutR to tis binding site.
Glucitol induction in Bacillus subtilis requires a transcription activator, GutR, and a sequence located upstream of the gut promoter. To understand the initial steps involved in the GutR-mediated transcription activation process and the physiological roles of glucitol, GutR was overproduced and purified. In the absence of glucitol, GutR exists as a monomer and binds directly to its binding site in the gut regulatory region. This binding site was mapped to a 29-base pair imperfect inverted repeat located between -78 and -50, and there is only one GutR binding site within the regulatory region. The kinetic parameters of the interaction between GutR and its binding site were monitored in real time using surface plasmon resonance. The half-life of the GutR-DNA complex in the absence of glucitol was estimated to be 6.8 min. In contrast, in the presence of glucitol, the half-life of the complex was extended to longer than 19 h by affecting only the off-rate but not the on-rate. This effect is glucitol-specific. These data indicate that glucitol binds to GutR and induces GutR to have an extremely tight binding at its binding site. The physiological relevance of this process in transcription activation is discussed.
Poon
K K
KK
Division of Cellular, Molecular and Microbial Biology, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Chen
C L
CL
Wong
S L
SL
D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D019921:Biotinylation; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004234:Diuretics, Osmotic / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000378:metabolism*; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013012:Sorbitol / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004234:Diuretics, Osmotic; C087480:GutR protein, Bacillus subtilis; D007202:Indicators and Reagents; D013012:Sorbitol; D004247:DNA; D019809:Streptavidin