title: 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.
abstract: The synechococcal metallothionein locus smt consists of two divergent genes: smtA coding for the metallothionein SmtA, and smtB coding for the trans-acting 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 operator/promoter DNA and to the separate S1/S2 and S3/S4 half-operator oligonucleotides using sedimentation equilibrium and sedimentation velocity measurements. 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 SmtB, forms a S1/S2-SmtB:SmtB-S3/S4 bridge complex. Combining these results with previously published binding interference data, which showed consecutive S1/S2 and S3/S4 SmtB occupancy on the operator/promoter DNA, we have developed a model for the establishment of the repression complex that appears to 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 considerably in the presence of Zn2+. Hence, SmtB remains bound to the operator sites when Zn2+ ions are present. This result is further supported by gel retardation assay. Failure of the metal ions to dissociate SmtB from the DNA points to a hitherto unknown function of SmtB in the regulation of the smt locus.
authors.affiliation: Graduate Program in Biophysical Sciences, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: S R
authors.initials: SR
authors.lastname: Kar
authors.forename: J
authors.initials: J
authors.lastname: Lebowitz
authors.forename: S
authors.initials: S
authors.lastname: Blume
authors.forename: K B
authors.initials: KB
authors.lastname: Taylor
authors.forename: L M
authors.initials: LM
authors.lastname: Hall
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D000458:Cyanobacteria / Q000201:enzymology; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D066298:In Vitro Techniques; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D009875:Operator Regions, Genetic / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000502:physiology; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D017384:Sequence Deletion; D014461:Ultracentrifugation; D015032:Zinc / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D012097:Repressor Proteins; D004247:DNA; D008668:Metallothionein; D015032:Zinc