Lee Sung-Jae, Surma Melanie, Seitz Sabine, Hausner Winfried, Thomm Michael, Boos Winfried
Department of Biology, University of Konstanz, 78457 Konstanz, Germany.
Mol Microbiol. 2007 Jul;65(2):305-18. doi: 10.1111/j.1365-2958.2007.05780.x. Epub 2007 Jun 21.
The characterization of the transcriptional regulator TrmBL1 of the hyperthermophilic archaeon Pyrococcus furiosus, homologous to TrmB (transcriptional regulator of the maltose system), was studied. The genome of P. furiosus contains three TrmB paralogues. One of the TrmB-like proteins (TrmBL), PF0124 (TrmBL1), was analysed in more detail. It regulated the expression of the genes encoding enzymes of the glycolytic pathway as well as the maltodextrin (MD) ABC transporter. By molecular sieve chromatography, purified TrmBL1 behaved at ambient temperature as a tetramer of 148.8 kDa. In the presence of 1 mM maltotriose or 5 mM maltose TrmBL1 formed octamers. As shown by electrophoretic mobility shift assay (EMSA) TrmBL1 was found to bind the MD (maltodextrin ABC transport genes) promoter DNA with sixfold higher binding affinity (K(d) 0.2 microM) than to the trehalose/maltose ABC transporter (TM) promoter (K(d) 1.2 microM). Maltotriose and maltose interfered in these assays indicating inducer function. In vitro transcription assays using purified transcription components corroborated the data obtained with EMSA and showed inhibition of transcription of the MD promoter by TrmBL1. Recently, van de Werken et al. (FEMS Microbiol Lett 2006; 260: 69-76) identified TGM, a conserved sequence (Thermococcales-Glycolytic-Motif) upstream of genes encoding glycolytic enzymes and the MD ABC transporter. The position of TGM is invariably located downstream of the BRE-TATA box and overlapping the transcription start site on each promoter. By footprint analysis TrmBL1 was found to recognize the TGM sequence in several TGM-containing promoter sequences. We identified the recognition helix in TrmBL1 revealing tyrosine (Y49) to be essential for target DNA binding. However, the TGM motif was not essential for TrmBL1 binding. We conclude that TrmBL1 is a global sugar-sensing transcriptional regulator controlling the genes of transport systems and of sugar-metabolizing enzymes.
对嗜热古菌激烈火球菌（Pyrococcus furiosus）中与麦芽糖系统转录调节因子TrmB同源的转录调节因子TrmBL1进行了表征研究。激烈火球菌的基因组包含三个TrmB旁系同源物。对其中一个类TrmB蛋白（TrmBL），即PF0124（TrmBL1）进行了更详细的分析。它调节糖酵解途径中编码酶的基因以及麦芽糖糊精（MD）ABC转运蛋白的表达。通过分子筛色谱法，纯化后的TrmBL1在环境温度下表现为148.8 kDa的四聚体。在存在1 mM麦芽三糖或5 mM麦芽糖的情况下，TrmBL1形成八聚体。如电泳迁移率变动分析（EMSA）所示，发现TrmBL1与MD（麦芽糖糊精ABC转运基因）启动子DNA结合的亲和力（K(d) 0.2 microM）比与海藻糖/麦芽糖ABC转运蛋白（TM）启动子结合的亲和力（K(d) 1.2 microM）高六倍。麦芽三糖和麦芽糖在这些分析中产生干扰，表明具有诱导功能。使用纯化的转录成分进行的体外转录分析证实了用EMSA获得的数据，并显示TrmBL1抑制MD启动子的转录。最近，范德韦肯等人（FEMS Microbiol Lett 2006; 260: 69 - 76）鉴定了TGM，这是编码糖酵解酶和MD ABC转运蛋白的基因上游的一个保守序列（嗜热栖热菌属 - 糖酵解 - 基序）。TGM的位置始终位于BRE - TATA框下游，且与每个启动子上的转录起始位点重叠。通过足迹分析发现TrmBL1能识别几个含TGM的启动子序列中的TGM序列。我们确定了TrmBL1中的识别螺旋，发现酪氨酸（Y49）对于靶DNA结合至关重要。然而，TGM基序对于TrmBL1结合并非必不可少。我们得出结论，TrmBL1是一种全局糖感应转录调节因子，控制转运系统和糖代谢酶的基因。
