SmtB-DNA and protein-protein interactions in the formation of the cyanobacterial metallothionein repression complex: Zn2+ does not dissociate the protein-DNA complex in vitro

The synechococcal metallothionein locus smt consists of two divergent genes : smtA coding for the metallothionein SmtA, and smtB coding for the trans-a cting regulator SmtB. The latter binds at two inverted repeats, designated S1/S2 and S3/S4, in the overlapping promoter/operator sites between the two genes. We have determined the binding stoichiometries to the entire operat or/promoter DNA and to the separate S1/S2 and S3/S4 half-operator oligonucl etides using sedimentation equilibrium and sedimentation velocity measureme nts. The full promoter/operator DNA binds two SmtB dimers. The hydrodynamic behavior of this complex supports a compact nucleoprotein structure. Each separate S1/S2 and S3/S4 operator sequence also binds two dimers. An equal molar mixture of separate S1/S2 and S3/S4 operator sequences, in excess Smt B, forms a S1/S2-SmtB:SmtB-S3/S4 bridge complex. Combining these results wi th previously published binding interference data, which showed consecutive S1/S2 and S3/S4 SmtB occupancy on the operator/promoter DNA, we have devel oped a model for the establishment of the repression complex that appears t o involve significant DNA compaction, presumably DNA bending, stabilized by SmtB-SmtB bridge interactions. DNase I footprinting titrations also showed consecutive S1/S2 and S3/S4 SmtB occupancy. The footprints expand consider ably in the presence of Zn2+. Hence, SmtB remains bound to the operator sit es when Zn2+ ions are present. This result is further supported by gel reta rdation assay. Failure of the metal ions to dissociate SmtB from the DNA po ints to a hitherto unknown function of SmtB in the regulation of the smt lo cus.