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Missense mutations in the Bacillus subtilis gnt repressor that diminish operator binding ability
1Department of Biotechnology, Faculty of Engineering, Fukuyama University, Japan.
Journal of Molecular Biology
|May 20, 1993
Summary
The Bacillus subtilis GntR repressor regulates the gnt operon. Mutations in its N-terminal region weaken DNA binding, revealing key residues for operator interaction.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The Bacillus subtilis gnt operon controls gluconate metabolism.
- This operon is negatively regulated by the GntR repressor protein.
- GntR belongs to a distinct family of bacterial regulatory proteins.
Purpose of the Study:
- To identify the DNA-binding domain of the GntR protein.
- To understand the role of specific amino acid residues in GntR's operator binding ability.
Main Methods:
- Hydroxylamine mutagenesis was used to generate GntR mutants.
- Sequence analysis identified amino acid substitutions in mutant proteins.
- In vivo and in vitro assays, including gel retardation, assessed operator binding affinity.
Main Results:
- Mutations GntR43L, GntR66T, GntR74K, and GntR75Q, located in the N-terminal conserved region, diminished operator binding.
- Operator binding affinity decreased in the order: wild-type GntR > GntR66T > GntR75Q > GntR74K > GntR43L.
- Equilibrium dissociation constants (Kd) for wild-type, GntR66T, GntR75Q, and GntR74K were 0.43, 2.6, 4.2, and 8.8 x 10(-10) M, respectively.
Conclusions:
- The N-terminal conserved region of GntR is crucial for its DNA-binding activity.
- Specific amino acid residues within this region directly impact GntR's interaction with its operator DNA.
- This study elucidates the molecular basis of GntR-mediated transcriptional regulation in Bacillus subtilis.