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.
Biochemistry
; 40(44): 13378-89, 2001 Nov 06.
Article
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| MEDLINE
| ID: mdl-11683648
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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Represoras
/
Proteínas Bacterianas
/
ADN
/
Proteínas de Unión al ADN
/
Metalotioneína
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Biochemistry
Año:
2001
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos