Gao J, Gussin G N
Department of Biology, University of Iowa, Iowa City 52242.
EMBO J. 1991 Dec;10(13):4137-44. doi: 10.1002/j.1460-2075.1991.tb04991.x.
In vitro, Pseudomonas aeruginosa TrpI protein activates transcription initiation at the trpBA promoter (trpPB) and represses initiation at its own promoter (trpPI), which diverges from, and overlaps, trpPB. Indoleglycerol phosphate (InGP) reduces the TrpI concentration required for binding to its strong binding site (site I), as measured by repression of trpPI; it also facilitates activation of trpPB, presumably because it enables TrpI to bind to a weaker binding site (site II) and thereby interact with RNA polymerase. The role of site II and InGP in regulation of the two promoters was investigated by constructing site II mutants. A 2 bp substitution affected the ability of TrpI to activate trpPB, but did not significantly affect TrpI binding to site II. A more extensive (8 bp) substitution inhibited TrpI-mediated activation of trpPB and TrpI-mediated protection of site II in a DNase I footprinting assay. However, the mutation did not alter the pattern of TrpI binding observed in gel retardation experiments. In particular, a more slowly-migrating complex (Complex 2) whose appearance was correlated with TrpI binding to site II was formed equally well on a wild-type or substituted DNA fragment. Based on the mutant phenotypes, we propose that a particular sequence of protein--protein and protein--DNA interactions is required for activation of trpPB by TrpI and InGP.
在体外，铜绿假单胞菌TrpI蛋白可激活色氨酸操纵子启动子（trpPB）处的转录起始，并抑制其自身启动子（trpPI）处的转录起始，trpPI与trpPB方向相反且部分重叠。磷酸吲哚甘油（InGP）可降低TrpI与其强结合位点（位点I）结合所需的浓度，这可通过trpPI的抑制作用来衡量；它还促进trpPB的激活，可能是因为它能使TrpI与较弱的结合位点（位点II）结合，从而与RNA聚合酶相互作用。通过构建位点II突变体研究了位点II和InGP在两个启动子调控中的作用。2个碱基对的替换影响了TrpI激活trpPB的能力，但对TrpI与位点II的结合没有显著影响。在DNA酶I足迹实验中，更广泛的（8个碱基对）替换抑制了TrpI介导的trpPB激活和TrpI介导的位点II保护。然而，该突变并未改变凝胶阻滞实验中观察到的TrpI结合模式。特别是，一种迁移较慢的复合物（复合物2）的出现与TrpI与位点II的结合相关，在野生型或替换的DNA片段上形成的情况相同。基于突变体表型，我们提出TrpI和InGP激活trpPB需要特定的蛋白质-蛋白质和蛋白质-DNA相互作用序列。
