Brinkman A B, Dahlke I, Tuininga J E, Lammers T, Dumay V, de Heus E, Lebbink J H, Thomm M, de Vos W M, van Der Oost J
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands.
J Biol Chem. 2000 Dec 8;275(49):38160-9. doi: 10.1074/jbc.M005916200.
The archaeal transcriptional initiation machinery closely resembles core elements of the eukaryal polymerase II system. However, apart from the established basal archaeal transcription system, little is known about the modulation of gene expression in archaea. At present, no obvious eukaryal-like transcriptional regulators have been identified in archaea. Instead, we have previously isolated an archaeal gene, the Pyrococcus furiosus lrpA, that potentially encodes a bacterial-like transcriptional regulator. In the present study, we have for the first time addressed the actual involvement of an archaeal Lrp homologue in transcription modulation. For that purpose, we have produced LrpA in Escherichia coli. In a cell-free P. furiosus transcription system we used wild-type and mutated lrpA promoter fragments to demonstrate that the purified LrpA negatively regulates its own transcription. In addition, gel retardation analyses revealed a single protein-DNA complex, in which LrpA appeared to be present in (at least) a tetrameric conformation. The location of the LrpA binding site was further identified by DNaseI and hydroxyl radical footprinting, indicating that LrpA binds to a 46-base pair sequence that overlaps the transcriptional start site of its own promoter. The molecular basis of the transcription inhibition by LrpA is discussed.
古菌的转录起始机制与真核生物聚合酶II系统的核心元件极为相似。然而，除了已确定的基础古菌转录系统外，对于古菌中基因表达的调控了解甚少。目前，尚未在古菌中鉴定出明显的类似真核生物的转录调节因子。相反，我们之前分离出了一个古菌基因，即嗜热栖热菌的lrpA，它可能编码一种类似细菌的转录调节因子。在本研究中，我们首次探讨了古菌Lrp同源物在转录调控中的实际作用。为此，我们在大肠杆菌中表达了LrpA。在无细胞的嗜热栖热菌转录系统中，我们使用野生型和突变的lrpA启动子片段来证明纯化的LrpA对其自身转录具有负调控作用。此外，凝胶阻滞分析揭示了一个单一的蛋白质-DNA复合物，其中LrpA似乎以（至少）四聚体构象存在。通过DNaseI和羟基自由基足迹法进一步确定了LrpA结合位点的位置，表明LrpA与一个46碱基对的序列结合，该序列与其自身启动子的转录起始位点重叠。本文还讨论了LrpA抑制转录的分子基础。
