Lee Sung-Jae, Engelmann Afra, Horlacher Reinhold, Qu Qiuhao, Vierke Gudrun, Hebbeln Carina, Thomm Michael, Boos Winfried
Department of Biology, University of Konstanz, Germany.
J Biol Chem. 2003 Jan 10;278(2):983-90. doi: 10.1074/jbc.M210236200. Epub 2002 Nov 7.
We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 microm and 0.5 mm sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80 degrees C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.
我们报道了TrmB的特性，它是一种表观分子量为38,800的蛋白质，参与嗜热栖热菌中一个基因簇（malE malF malG orf trmB malK）的麦芽糖特异性调控，该基因簇编码一种依赖结合蛋白的海藻糖和麦芽糖ABC转运蛋白。TrmB分别在20微摩尔和0.5毫摩尔糖浓度下与麦芽糖和海藻糖半最大结合。麦芽糖而非海藻糖的结合显示出S形迹象，并使内在色氨酸荧光猝灭15%，表明麦芽糖结合时发生了构象变化。TrmB会导致含有通过足迹法鉴定的启动子和上游调控基序的DNA片段的电泳迁移率发生改变。TrmB引起的条带迁移可被麦芽糖阻止。已建立了使用来自激烈火球菌纯化成分的体外转录分析，以显示在80℃下pmalE启动子依赖性转录。TrmB特异性抑制转录，且这种抑制作用可被麦芽糖和海藻糖抵消。这些数据表明TrmB是嗜热栖热菌海藻糖/麦芽糖转运操纵子的麦芽糖特异性阻遏物。
