Elucidation of gene regulation systems based on genome analysis

我们发现了一个新的调控因子--热球菌糖酵解调节因子(TGR)，它在高温嗜热考古菌中既是转录的激活因子，又是转录的抑制因子。TGR(TK1769)与调节海藻糖/麦芽糖ABC转运蛋白基因的转录抑制因子(PF1743)有28%的相似性(28%相同)，但与P.fuiosus(PF0124)中的TRMB蛋白的同源性更高(67%)。TGR破碎菌(ΔTGR)在糖异生条件下的生长速度明显低于野生型菌株，而在糖酵解条件下的生长速度与野生型菌株相当。全基因组芯片分析表明，几乎所有与糖酵解和麦芽糊精代谢相关的基因在ΔTGR突变体中的转录水平都相对较高，即使在糖异生条件下也是如此。在这些条件下，ΔTGR突变体在糖异生基因的转录激活方面也表现出缺陷，表明TGR既具有激活功能，也具有抑制功能。受TGR调控的基因包含一个先前发现的序列基序，TGM(热球菌糖酵解基序)。TGM在糖异生启动子中位于BRE/TATA序列的上游，在糖酵解启动子中位于其下游。凝胶迁移率改变分析表明，重组TGR蛋白与含有TGM的启动子区域特异性结合。当加入麦芽三糖时，TGR从DNA中释放出来，这表明这种糖很可能是生理效应。我们的结果有力地表明，TGR是一个全球转录调节因子，通过与TGM的直接结合，同时控制Kodakaraensis的糖酵解和糖异生代谢。

We have identified a novel regulator, Tgr (Thermococcales glycolytic regulator), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD 1. Tgr (TK1769) displays similarity (28% identical) to Pyrococcus furiosus TrmB (PF1743), a transcriptional repressor regulating the trehalose/maltose ABC transporter genes, but is more closely related (67%) to a TrmB paralog in P.furiosus (PF0124). Growth of a tgr disruption strain (Δtgr) displayed a significant decrease in growth rate under gluconeogenic conditions compared to the wild-type strain, while comparable growth rates were observed under glycolytic conditions. A whole genome microarray analysis revealed that transcript levels of almost all genes related to glycolysis and maltodextrin metabolism were at relatively high levels in the Δtgr mutant even under gluconeogenic conditions. The Δtgr mutant also displayed defects in the transcriptional activation of gluconeogenic genes under these conditions, indicating that Tgr functions as both an activator and a repressor. Genes regulated by Tgr contain a previously identified sequence motif, TGM (Thermococcales glycolytic motif). The TGM was positioned upstream of the BRE/TATA sequence in gluconeogenic promoters, and downstream of it in glycolytic promoters. Electrophoretic mobility shift assay indicated that recombinant Tgr protein specifically binds to promoter regions containing a TGM. Tgr was released from the DNA when maltotriose was added, suggesting that this sugar is most likely the physiological effector. Our results strongly suggest that Tgr is a global transcriptional regulator that simultaneously controls, in response to sugar availability, both glycolytic and gluconeogenic metabolism in T.kodakaraensis via its direct binding to the TGM.