Ikeda Yu, Minoshima Hikari, Satoh Mai, Ishikawa Tomoaki, Kawashima-Ohya Yoshie, Tomobe Koji, Omata Yoshiaki, Kawashima Tsuyoshi
Department of Molecular Biology, Faculty of Pharmaceutical Science, Yokohama College of Pharmacy, Yokohama 245-0066, Japan.
J Gen Appl Microbiol. 2012;58(6):465-73. doi: 10.2323/jgam.58.465.
Because archaea possess many respiratory enzymes or radical scavengers with catalytic domains that contain iron, the expression of the genes encoding these enzymes might be regulated by iron acquisition. The genome of an archaeon, Thermoplasma volcanium contains a gene that encodes Fur (TVN0292). The fur gene of T. volcanium was amplified by PCR, and cloned into plasmid pET28a. TvFur (T. volcanium Fur protein) was expressed in E. coli cells and then purified. EMSA revealed that TvFur binds to its own promoter DNA. The binding to its own promoter was in an Mn(2+)-, Zn(2+)-, and Ni(2+)-dependent manner. DNase I footprinting analysis revealed that the binding sequence of tvfur promoter was 5'-G TTATTAT G TTTATAT A TTAATTA G-3'. An analysis utilizing oligonucleotides in TvFur-binding sequences revealed that TvFur binds to the TATA-box or regions in the vicinity of the TATA-box in the promoter. These results indicated that TvFur regulates transcription depending on the availability of environmental divalent cations.
由于古菌拥有许多呼吸酶或具有含铁催化结构域的自由基清除剂，编码这些酶的基因表达可能受铁摄取的调控。嗜热栖热菌（Thermoplasma volcanium）的基因组包含一个编码Fur（TVN0292）的基因。通过PCR扩增嗜热栖热菌的fur基因，并克隆到质粒pET28a中。TvFur（嗜热栖热菌Fur蛋白）在大肠杆菌细胞中表达，然后进行纯化。电泳迁移率变动分析（EMSA）表明TvFur与其自身的启动子DNA结合。与自身启动子的结合以Mn(2+)、Zn(2+)和Ni(2+)依赖的方式进行。DNase I足迹分析表明tvfur启动子的结合序列为5'-G TTATTAT G TTTATAT A TTAATTA G-3'。利用TvFur结合序列中的寡核苷酸进行的分析表明，TvFur与启动子中的TATA盒或TATA盒附近区域结合。这些结果表明，TvFur根据环境二价阳离子的可用性调节转录。
