Chang M, Crawford I P
Department of Microbiology, University of Iowa, Iowa City 52242.
J Bacteriol. 1991 Mar;173(5):1590-7. doi: 10.1128/jb.173.5.1590-1597.1991.
Expression of the trpBA gene pair of Pseudomonas aeruginosa is regulated by the endogenous level of indoleglycerol phosphate (InGP) and the trpI gene product. The TrpI protein binds to the -77 to -32 region of the trpBA promoter. This region is divisible into two sites: site I, which is protected by TrpI in the presence and absence of InGP; and site II, which is protected by TrpI only in the presence of InGP. Recently, the trpI gene was subcloned into an expression vector and the protein was overproduced in Escherichia coli. The TrpI protein was purified to 80 to 95% purity. The molecular weight of native TrpI protein is estimated to be 129,000 by gel exclusion chromatography, and therefore it is likely a tetramer composed of 31,000-dalton monomers. Gel retardation assays with the purified TrpI protein demonstrated that InGP increases the affinity of TrpI for sites I and II approximately 17- and 14-fold, respectively. Binding of TrpI to site I is site II independent. However, the protein has low intrinsic affinity for site II and its binding to site II is site I dependent. Therefore, binding of TrpI to site II probably requires its interaction with a second TrpI molecule at site I.
铜绿假单胞菌trpBA基因对的表达受磷酸吲哚甘油（InGP）的内源水平和trpI基因产物的调控。TrpI蛋白与trpBA启动子的-77至-32区域结合。该区域可分为两个位点：位点I，在有或没有InGP的情况下都受TrpI保护；位点II，仅在有InGP的情况下受TrpI保护。最近，trpI基因被亚克隆到一个表达载体中，并在大肠杆菌中过量表达该蛋白。TrpI蛋白被纯化至80%至95%的纯度。通过凝胶排阻色谱法估计天然TrpI蛋白的分子量为129,000，因此它可能是由31,000道尔顿单体组成的四聚体。用纯化的TrpI蛋白进行的凝胶阻滞试验表明，InGP分别使TrpI对位点I和位点II的亲和力增加约17倍和14倍。TrpI与位点I的结合不依赖于位点II。然而，该蛋白对位点II的固有亲和力较低，其与位点II的结合依赖于位点I。因此，TrpI与位点II的结合可能需要其与位点I上的第二个TrpI分子相互作用。
