Missense mutations in the Bacillus subtilis gnt repressor that diminish operator binding ability.
The Bacillus subtilis gnt operon is negatively regulated by the gnt repressor (GntR, 243 amino acids), which is antagonized by gluconate. The GntR protein belongs to a new family of bacterial regulatory proteins (GntR family). To locate the DNA-binding domain of the GntR protein, we obtained mutations of this protein, by hydroxylamine mutagenesis, which diminish its operator binding ability. Sequence analysis of these mutations indicated that the mutant GntR proteins (GntR43L, GntR66T, GntR74K and GntR75Q) had amino acid substitutions (Ser43 to Leu, Ala66 to Thr, Glu74 to Lys and Arg75 to Gln), respectively. They were all located within the N-terminal conserved region of the GntR family. In vivo and in vitro analysis of these GntR proteins indicated that their relative operator binding abilities became weaker in the order of GntR (wild type), GntR66T, GntR75Q, GntR74K and GntR43L. The equilibrium dissociation constants of GntR (wild type), GntR66T, GntR75Q and GntR74K as to operator binding were determined by gel retardation assays to be 0.43, 2.6, 4.2 and 8.8 M x 10(-10), respectively.
Department of Biotechnology, Faculty of Engineering, Fukuyama University, Japan.
K
K
Yoshida
Y
Y
Fujita
A
A
Sarai
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D009875:Operator Regions, Genetic / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C081598:GntR protein, E coli; D012097:Repressor Proteins; D014157:Transcription Factors