COOPERATIVE BINDING OF THE ESCHERICHIA-COLI REPRESSOR OF BIOTIN BIOSYNTHESIS TO THE BIOTIN OPERATOR SEQUENCE

Regulation of biotin biosynthesis and retention in Escherichia coli de pends on a complex set of coupled protein-protein, protein-nucleic aci d, and protein-small molecule interactions. The complexity of the biot in system is analogous to that found in gene regulatory systems from o ther prokaryotes and from eukaryotes. Quantitative understanding of th ese systems requires thermodynamic studies of the individual contribut ing interactions. We have initiated such studies of the biotin regulat ory interactions. The assembly states of the biotin operon repressor ( BirA) and its complex with the allosteric effector, bio-5'-AMP, have b een determined by analytical gel filtration chromatography. Both the a po- and holo-repressors are monomeric at protein concentrations severa l orders of magnitude higher than those required for DNA binding. Resu lts 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 measu red using the quantitative DNase footprint technique. Analysis of the data indicates that the binding process is best described by a coopera tive model. An upper limit for the cooperative free energy is estimate d to be between -2.0 and -3.0 kcal/mol.