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Bacillus subtilis CodY operators contain overlapping CodY binding sites
1Department of Microbiology, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118-2526, USA.
Journal of Bacteriology
|July 19, 2011
Summary
Bacillus subtilis CodY protein binds DNA more effectively at pH 6.0 than pH 8.0. This pH-dependent DNA binding influences gene regulation by CodY, a global transcriptional regulator activated by amino acids.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- CodY is a global transcriptional regulator in bacteria, activated by branched-chain amino acids.
- CodY recognizes a specific 15-bp palindromic DNA motif (AATTTTCNGAAAATT).
Purpose of the Study:
- To investigate the effect of pH on the DNA binding affinity and regulatory function of Bacillus subtilis CodY.
- To characterize the specific DNA sequences and binding mechanisms of CodY at different pH levels.
Main Methods:
- Gel mobility shift assays were employed to assess CodY's DNA binding affinity at varying pH.
- DNase I footprinting was utilized to identify CodY-protected DNA regions within target promoters.
- Mutational analysis of promoter regions was performed to confirm the role of identified motifs in CodY regulation.
Main Results:
- Bacillus subtilis CodY exhibits higher DNA binding affinity at pH 6.0 compared to pH 8.0.
- Isoleucine activation of CodY is enhanced at pH 6.0, with reduced nonspecific DNA binding.
- CodY binding sites within the hutPp and ureAp(3) promoters contain overlapping copies of the 15-bp motif.
Conclusions:
- pH significantly influences the DNA binding characteristics and regulatory activity of Bacillus subtilis CodY.
- Overlapping sequence motifs are crucial for CodY binding to native operator sites, suggesting a complex regulatory mechanism.
- These findings provide insights into the environmental factors affecting bacterial gene expression regulated by CodY.
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