Predicting metals sensed by ArsR-SmtB repressors: allosteric interference by a non-effector metal
Predicting metals sensed by ArsR-SmtB repressors: allosteric interference by a non-effector metal
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DOI：
10.1111/j.1365-2958.2006.05029.x
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发表时间：
2006-02-01
影响因子：
3.6
通讯作者：
Robinson, NJ
中科院分区：
文献类型：
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作者：
Harvie, DR;Andreini, C;Robinson, NJ
许多细菌基因组编码多个金属敏感的ArsR-SmtB转录抑制因子。人们有兴趣了解和预测它们的金属特性。在这里，我们分析了枯草芽孢杆菌的两个arsR-smtB基因，ydeT和yoza(现为Aser和czrA)。纯化的Aser和CHARA在凝胶滞留和荧光各向异性分析中与Delta Aser和Delta czrA细胞中去抑制的启动子片段形成复合体。预测候选的(I)部分硫代、α3-螺旋(Aser)和(Ii)四面体、非硫代、α5-螺旋(ChrA)金属结合部位，然后在体外和/或体内进行测试。这类网站的先例是感知亚砷酸盐/锑矿(阿尔法3)和锌(阿尔法5)。这与Aser和ChurA各自的金属诱导剂抑制枯草杆菌中的启动子有关，并与在体外阻碍蛋白质-DNA复合体形成的金属相匹配。报道了1024个ArsR-SmtB同系物的可能感觉位点。虽然Aser在体内没有感觉到锌，但它在体外利用α3硫醇与锌结合，但不影响Aser DNA的结合。如果选择性依赖于变构的歧视性触发，而不仅仅是选择性的金属结合，那么紧密的非效应性金属络合物理论上可能会抑制金属传感。在体外，Aser在等摩尔锌中对亚砷酸盐敏感，而在等摩尔亚砷酸盐中则对锌敏感。然而，铜离子并不影响CHARA-DNA络合物的形成，但在体外确实抑制了锌介导的变构并阻止了锌的结合。体内对铜的获取必须加以控制，以避免铜的形成。
Many bacterial genomes encode multiple metal-sensing ArsR-SmtB transcriptional repressors. There is interest in understanding and predicting their metal specificities. Here we analyse two arsR-smtB genes, ydeT and yozA (now aseR and czrA) from Bacillus subtilis. Purified AseR and CzrA formed complexes in gel-retardation and fluorescence-anisotropy assays with fragments of promoters that were derepressed in Delta aseR and Delta czrA cells. Candidate (i) partly thiolate, alpha 3-helix (for AseR) and (ii) tetrahedral, non-thiolate, alpha 5-helix (for CzrA) metal binding sites were predicted then tested in vitro and/or in vivo. The precedents are for such sites to sense arsenite/antimonite (alpha 3) and zinc (alpha 5). This correlated with the respective metal inducers of AseR and CzrA repressed promoters in B. subtilis and matched the metals that impaired formation of protein-DNA complexes in vitro. The putative sensory sites of 1024 ArsR-SmtB homologues are reported. Although AseR did not sense zinc in vivo, it bound zinc in vitro exploiting alpha 3 thiols, but AseR DNA binding was not impaired by zinc. If selectivity relies on discriminatory triggering of allostery not just selective metal binding, then tight non-effector metal complexes could theoretically inhibit metal sensing. AseR remained arsenite-sensitive in equimolar zinc, while CzrA remained zinc-sensitive in equimolar arsenite in vitro. However, cupric ions did not impair CzrA-DNA complex formation but did inhibit zinc-mediated allostery in vitro and prevent zinc binding. Access to copper must be controlled in vivo to avoid formation of cupric CzrA.