Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, OH 45056, United States.
J Inorg Biochem. 2012 Jun;111:164-72. doi: 10.1016/j.jinorgbio.2011.11.022. Epub 2011 Dec 2.
RT-PCR and DNA microarrays were used to probe for Zn(II)-responsive genes in E. coli cells that were made Zn(II) deficient. Microarray data revealed 114 genes were significantly up-regulated and 146 genes were significantly down-regulated in Zn(II) deficient conditions. The three most up-regulated genes were (1) znuA, which encodes for a periplasmic protein known to be involved with Zn(II) import, (2) yodA, which encodes for a periplasmic protein with unknown function, and (3) ykgM, which encodes for a ribosomal protein that is thought to be a paralog of ribosomal protein L31. YodA was over-expressed and purified as a maltose binding protein (MBP) fusion protein and shown to tightly bind 4 equivalents of Zn(II). Metal analyses showed that MBP-YkgM does not bind Zn(II). On the other hand, MBP-L31 tightly binds 1 equivalent of Zn(II). EXAFS studies on MBP-L31 suggest a ligand field of 1 histidine, 1 cysteine, and 2 additional N/O scatterers. Site-directed mutagenesis studies suggest that Cys16 coordinates Zn(II) in MBP-L31 and that the other three cysteines do not bind metal. These results are discussed in light of Zn(II) starvation model that has been postulated for B. subtilis.
RT-PCR 和 DNA 微阵列被用于探测在制造缺锌条件下的大肠杆菌细胞中对锌（II）有反应的基因。微阵列数据显示，在缺锌条件下有 114 个基因显著上调，146 个基因显著下调。上调最明显的三个基因是：(1) znuA，编码一种已知与锌（II）导入有关的周质蛋白；(2) yodA，编码一种功能未知的周质蛋白；(3) ykgM，编码一种核糖体蛋白，被认为是核糖体蛋白 L31 的同源物。YodA 被过表达并作为麦芽糖结合蛋白（MBP）融合蛋白纯化，并被证明能紧密结合 4 当量的锌（II）。金属分析表明，MBP-YkgM 不结合锌（II）。另一方面，MBP-L31 紧密结合 1 当量的锌（II）。对 MBP-L31 的 EXAFS 研究表明，配体场由 1 个组氨酸、1 个半胱氨酸和 2 个额外的 N/O 散射体组成。定点突变研究表明，Cys16 在 MBP-L31 中配位锌（II），而其他三个半胱氨酸不结合金属。这些结果与枯草芽孢杆菌提出的锌（II）饥饿模型进行了讨论。
