Molecular characterization of the transition state regulator AbrB from Bacillus stearothermophilus.
The Bacillus subtilis transition state regulator AbrB(su) is a DNA-binding protein that acts on several genes either as activator, repressor, or preventer. However, among genes under its control, neither common binding sites could be identified nor could the structural features of this broad and specific interaction be elucidated. Attempts to elucidate these interesting features by crystallizing AbrB(su) have failed so far. Therefore, to solve this problem, we focused in this work on identifying an AbrB(su) homologue from Bacillus stearothermophilus. Using a novel method, the entire abrB(st) gene of B. stearothermophilus was cloned and sequenced. The gene encodes a 95 amino acid protein that shows 77% identity and 85% similarity to the mesophilic B. subtilis protein. A calmodulin binding peptide-tagged fusion of the thermophilic gene was constructed for overexpression and efficient affinity column purification of the AbrB(st) protein. The purified protein showed, after removal of the tag, an oligomerization behavior through hexamer formation that is essential for its DNA binding activity.
Klein
W
W
Philipps Universität Marburg, Biochemie-FB Chemie, Hans-Meerwein-Strasse, D-35032, Marburg, Germany.
Winkelmann
D
D
Hahn
M
M
Weber
T
T
Marahiel
M A
MA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002148:Calmodulin-Binding Proteins / Q000737:chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
C060762:AbrB protein, Bacillus subtilis; D001426:Bacterial Proteins; D002148:Calmodulin-Binding Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA