Harvie Duncan R, Andreini Claudia, Cavallaro Gabriele, Meng Wenmao, Connolly Bernard A, Yoshida Ken-ichi, Fujita Yasutaro, Harwood Colin R, Radford David S, Tottey Stephen, Cavet Jennifer S, Robinson Nigel J
Cell and Molecular Biosciences, Medical School, University of Newcastle, Newcastle NE2 4HH, UK.
Mol Microbiol. 2006 Feb;59(4):1341-56. doi: 10.1111/j.1365-2958.2006.05029.x.
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 DeltaaseR and DeltaczrA cells. Candidate (i) partly thiolate, alpha3-helix (for AseR) and (ii) tetrahedral, non-thiolate, alpha5-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 (alpha3) and zinc (alpha5). 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 alpha3 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.
许多细菌基因组编码多种金属感应型ArsR-SmtB转录阻遏物。人们对理解和预测它们的金属特异性很感兴趣。在此，我们分析了来自枯草芽孢杆菌的两个arsR-smtB基因，即ydeT和yozA（现分别为aseR和czrA）。在凝胶阻滞和荧光各向异性分析中，纯化的AseR和CzrA与在ΔaseR和ΔczrA细胞中去阻遏的启动子片段形成复合物。预测了候选的（i）部分硫醇盐型、α3螺旋（针对AseR）和（ii）四面体、非硫醇盐型、α5螺旋（针对CzrA）金属结合位点，然后在体外和/或体内进行了测试。此前的研究表明，此类位点可感应亚砷酸盐/锑酸盐（α3）和锌（α5）。这与枯草芽孢杆菌中AseR和CzrA阻遏启动子的各自金属诱导剂相关，并且与在体外损害蛋白质-DNA复合物形成的金属相匹配。报告了1024个ArsR-SmtB同源物的假定感应位点。尽管AseR在体内不感应锌，但它在体外利用α3硫醇结合锌，不过锌并未损害AseR与DNA的结合。如果选择性依赖于别构的特异性触发而不仅仅是选择性的金属结合，那么紧密的非效应器金属复合物理论上可能会抑制金属感应。在体外等摩尔锌存在的情况下，AseR对亚砷酸盐仍敏感，而在等摩尔亚砷酸盐存在时，CzrA对锌仍敏感。然而，铜离子并未损害CzrA与DNA复合物的形成，但在体外确实抑制了锌介导的别构作用并阻止了锌的结合。在体内必须控制铜的获取，以避免形成铜离子结合的CzrA。
