A MAP OF THE BIOTIN REPRESSOR-BIOTIN OPERATOR INTERFACE - BINDING OF A WINGED HELIX-TURN-HELIX PROTEIN DIMER TO A 40 BASE-PAIR SITE

The Escherichia cell biotin repressor is a member of the ''winged heli x-turn-helix'' class of site-specific DNA binding proteins. The protei n binds as a dimer to the 40 bp biotin operator sequence. Although the structure of the aporepressor has been solved by X-ray crystallograph ic techniques, no structure of the holorepressor-DNA complex is yet av ailable. In order to characterize the structural features of the bioti n repressor-biotin operator interface we have applied a number of solu tion techniques including DNase I, hydroxyl radical and dimethyl sulfa te footprinting and the circular permutation or ''bending'' assay. Res ults of these combined studies indicate that each repressor monomer fo rms a bipartite interface with each half-site of the biotin operator s equence. The results imply that, in addition to the helix-turn-helix m odule of each monomer, a second structural element participates in the protein-DNA interface, The two bipartite protein-DNA interfaces appea r, moreover, to primarily involve the two 12 bp termini of the operato r site. Results of combined DNase I footprinting and circular permutat ion analysis indicate, furthermore, that the central 16 bp region that links the two termini becomes distorted concomitant with binding of h oloBirA. (C) 1998 Academic Press Limited.