Macromolecular assembly of the transition state regulator AbrB in its unbound and complexed states probed by microelectrospray ionization mass spectrometry.
The Bacillus subtilis global transition-state regulator AbrB specifically recognizes over 60 different DNA regulatory regions of genes expressed during cellular response to suboptimal environments. Most interestingly the DNA regions recognized by AbrB share no obvious consensus base sequence. To more clearly understand the functional aspects of AbrB activity, microelectrospray ionization mass spectrometry has been employed to resolve the macromolecular assembly of unbound and DNA-bound AbrB. Analysis of the N-terminal DNA binding domain of AbrB (AbrBN53, residues 1-53) demonstrates that AbrBN53 is a stable dimer, showing no apparent exchange with a monomeric form as a function of pH, ionic strength, solvent, or protein concentration. AbrBN53 demonstrates a capacity for DNA binding, underscoring the role of the N-terminal domain in both DNA recognition and dimerization. Full-length AbrB is shown to exist as a homotetramer. An investigation of the binding of AbrBN53 and AbrB to the natural DNA target element sinIR shows that AbrBN53 binds as a dimer and AbrB binds as a tetramer. This study represents the first detailed characterization of the stoichiometry of a transition-state regulator binding to one of its target promoters.
Biomedical Mass Spectrometry & Functional Proteomics Facility, Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA.
Linda M
LM
Benson
Jeffrey L
JL
Vaughn
Mark A
MA
Strauch
Benjamin G
BG
Bobay
Richele
R
Thompson
Stephen
S
Naylor
John
J
Cavanagh
D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli; D046911:Macromolecular Substances; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
C060762:AbrB protein, Bacillus subtilis; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014157:Transcription Factors; D004247:DNA