National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Biochem Biophys Res Commun. 2011 Apr 1;407(1):242-7. doi: 10.1016/j.bbrc.2011.03.011. Epub 2011 Mar 5.
The mechanism and target genes of regulation by Forkhead (FHA) domain-containing transcription factors have not yet been documented in Archaea. In this study, using a bacterial one-hybrid technique, we successfully screened and identified for the first time a target gene regulated by ST0829, an FHA domain-containing potential transcriptional factor in the hyperthermophilic archaeon Sulfolobus tokodaii. We show that ST0829 could specifically bind to the promoter region of ST2519p, the archaeal flagellar protein-encoding operon (including FlaG, FlaF, FlaH, FlaI, and FlaJ) by using both in vitro electrophoretic mobility shift assay and surface plasmon resonance experiments, and invivo chromatin immunoprecipitation assays. Furthermore, phosphorylation of the FHA domain-containing protein was found to negatively regulate its specific DNA-binding activity. The interaction between ST0829 and ST2519p could be inhibited by wild-type Ser/Thr protein kinase ST1565, but was not significantly affected by its mutant variant ST1565-K166A that lacks kinase activity. These findings not only increase our knowledge about the function of an archaeal FHA domain-containing regulator but also offer important insights for further understanding the signaling mechanism of environmental adaptation in archaea.
在古菌中，Forkhead（FHA）结构域含转录因子的调控机制和靶基因尚未有文献记载。在这项研究中，我们首次使用细菌单杂交技术，成功筛选并鉴定了嗜热古菌 Sulfolobus tokodaii 中一个 FHA 结构域含潜在转录因子 ST0829 调控的靶基因。我们通过体外电泳迁移率变动分析和表面等离子体共振实验以及体内染色质免疫沉淀实验，表明 ST0829 可以特异性地结合到 ST2519p 的启动子区域，ST2519p 编码的古菌鞭毛蛋白操纵子（包括 FlaG、FlaF、FlaH、FlaI 和 FlaJ）。此外，还发现 FHA 结构域含蛋白的磷酸化会负调控其特异性 DNA 结合活性。ST0829 和 ST2519p 之间的相互作用可以被野生型丝氨酸/苏氨酸蛋白激酶 ST1565 抑制，但对其缺乏激酶活性的突变变体 ST1565-K166A 没有显著影响。这些发现不仅增加了我们对古菌中一个 FHA 结构域含调控因子功能的认识，也为进一步了解古菌环境适应的信号机制提供了重要的见解。
