Coupling of protein assembly and DNA binding: biotin repressor dimerization precedes biotin operator binding.
The kinetics of coupling of protein dimerization and DNA binding have been investigated in the biotin repressor system. Two repressor monomers bind to the 40 base-pair biotin operator sequence. In previous analyses of equilibrium-binding data the weak dimerization of the repressor has justified using a model in which two protein monomers bind cooperatively to the operator site. Here, rapid kinetic methods have been used to directly determine the binding mechanism. Results of rapid-mixing DNaseI footprinting measurements of association of the repressor with operator indicate that the binding process involves at least two steps. Results of measurements of the unimolecular dissociation of the complex reveal a half-life of approximately 400 seconds. Analysis of the data using a combination of simulation and global non-linear least-squares analysis provides support for a binding model in which a preformed repressor dimer associates with the biotin operator. This kinetic model is consistent with the previously proposed model for regulation of the functional switch in the repressor from enzyme to site-specific DNA-binding protein.
Streaker
Emily D
ED
Department of Chemistry and Biochemistry, College of Life Sciences, University of Maryland, College Park, MD 20742-2021, USA.
Beckett
Dorothy
D
D001426:Bacterial Proteins / Q000378:metabolism*; D001710:Biotin / Q000235:genetics*; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D007700:Kinetics; D008957:Models, Genetic; D009876:Operon / Q000502:physiology*; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D001710:Biotin; D004247:DNA; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli