Erbe J L, Taylor K B, Hall L M
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 35294, USA.
Nucleic Acids Res. 1995 Jul 11;23(13):2472-8. doi: 10.1093/nar/23.13.2472.
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.
聚球藻PCC 7942的smtB基因编码一种金属调控的smtA基因的反式作用阻遏物，smtA基因编码一种II类金属硫蛋白。重组SmtB已在大肠杆菌中表达并纯化。使用重组SmtB或聚球藻PCC 6301的蛋白提取物进行的电泳迁移率变动分析揭示了与smt操纵子/启动子形成三种特异性复合物的浓度依赖性。SmtB也能够与金属直接相互作用，这通过65Zn与SmtB蛋白的结合以及在各种金属离子存在下阻遏物 - DNA复合物形成的抑制得以证明。对此类复合物的甲基化干扰分析确定了smt操纵子/启动子DNA内的四个蛋白质接触点。接触点似乎代表两对结合位点，在两个反向重复序列（核苷酸548 - 563、589 - 602）中的每一个中各有一对。每对中的接触点位于相对的DNA链上，相隔10个碱基对，使阻遏物结合位点位于DNA螺旋的相对两侧。基于电泳迁移率变动分析、甲基化干扰和分子大小计算，我们提出重组SmtB以多聚体方式结合到smt操纵子/启动子上。
