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The smtB gene of Synechococcus PCC 7942 encodes a trans-acting repressor of the metal-regulated smtA gene that encodes a class II metallothionein. Recombinant SmtB has been expressed in Escherichia coli and purified. Electrophoretic mobility shift assays using recombinant SmtB or a protein extract from Synechococcus PCC 6301 reveal the concentration-dependent formation of three specific complexes with the smt operator/promoter. SmtB is also capable of direct interaction with metals as evidenced by 65Zn binding to the SmtB protein as well as the inhibition of repressor-DNA complex formation in the presence of various metal ions. Methylation interference analysis of such complexes identifies four protein contact points within the smt operator/promoter DNA. The points of contact appear to represent two pairs of binding sites, one pair in each of two inverted repeats (nt 548-563, 589-602). The contact points within each pair lie on opposing DNA strands and are separated by 10 bp, placing the repressor binding sites on opposite sides of the DNA helix. Based on electrophoretic mobility shift assays, methylation interference and molecular size calculations we propose that recombinant SmtB binds to the smt operator/promoter in multimeric fashion.

The Synechococcus PCC7942 metallothionein repressor gene smtB has been cloned into a high expression vector and the protein purified to near homogeneity (>/=98%). Analytical ultracentrifugation studies demonstrate that the protein is predominantly dimeric in 0.1 M NaCl, pH 7.4, and 22 degrees C, exhibiting a monomer-dimer-tetramer equilibrium. The monomer-dimer (Ka(1,2)) and the dimer-tetramer (Ka(2,4)) association constants are 3.24 x 10(5) and 9.90 x 10(2) M-1, respectively. The repressor binds two Zn2+ ions per subunit with an overall Kd of 3.49 x 10(-6) M. In the presence of Zn2+, Ka(1, 2) increases by 2 orders of magnitude to 1.25 x 10(7) M-1 and the apparent weight-averaged sedimentation coefficient increases from 2. 00 to 2.22 S. The fact that the increase in sedimentation coefficient is greater than that predicted by increased dimerization is interpreted as caused by compaction of the structure in the presence of metal ions. At pH 6.0, 0.1 M NaCl, and 22 degrees C, the protein exhibits only a monomer-dimer equilibrium, with Ka(1,2) = 1.52 x 10(7) M-1 which is almost identical to that seen upon binding Zn2+ at pH 7.4. The compaction and conformational change in SmtB caused by Zn2+ is consistent with a role for this altered quaternary state in derepression of smtA in Synechococcus challenged with heavy metal ions.

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