Cooperative binding of the Escherichia coli repressor of biotin biosynthesis to the biotin operator sequence.
Regulation of biotin biosynthesis and retention in Escherichia coli depends on a complex set of coupled protein-protein, protein-nucleic acid, and protein-small molecule interactions. The complexity of the biotin system is analogous to that found in gene regulatory systems from other prokaryotes and from eukaryotes. Quantitative understanding of these systems requires thermodynamic studies of the individual contributing interactions. We have initiated such studies of the biotin regulatory interactions. The assembly states of the biotin operon repressor (BirA) and its complex with the allosteric effector, bio-5'-AMP, have been determined by analytical gel filtration chromatography. Both the apo- and holo-repressors are monomeric at protein concentrations several orders of magnitude higher than those required for DNA binding. Results of stoichiometric DNA binding measurements indicate that the BirA-biotin operator (bioO) complex consists of two holo-repressor monomers per operator site. Equilibrium binding of BirA to bioO has been measured using the quantitative DNase footprint technique. Analysis of the data indicates that the binding process is best described by a cooperative model. An upper limit for the cooperative free energy is estimated to be between -2.0 and -3.0 kcal/mol.
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore 21228.
J
J
Abbott
D
D
Beckett
D000227:Adenine Nucleotides; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009875:Operator Regions, Genetic; D011994:Recombinant Proteins; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors
C082099:5'-AMP-biotin; D000227:Adenine Nucleotides; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D000249:Adenosine Monophosphate; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli