Stauffer L T, Stauffer G V
Department of Microbiology, University of Iowa, Iowa City 52242.
J Bacteriol. 1994 Oct;176(20):6159-64. doi: 10.1128/jb.176.20.6159-6164.1994.
We constructed a set of deletions upstream of the gcv promoter and analyzed the effects of the deletions on expression of a gcvT-lacZ gene fusion. A deletion that ends at position -313 upstream of the transcription initiation site (+1) results in reduced levels of gcvT-lacZ expression, but the fusion is still inducible by glycine and repressible by purines. A deletion that ends at position -169 results in loss of both GcvA- and Lrp-mediated activation of the gcvT-lacZ fusion. The endpoints of delta -313 and delta -169 also define a site that down-regulates gcvT-lacZ expression two- to threefold. A deletion that ends at position -89 upstream from the transcription initiation site still shows PurR-mediated repression, suggesting that PurR-mediated repression is not by direct interference with the GcvA- and Lrp-mediated regulatory mechanism(s). Gel mobility shift assays and DNase I footprinting showed that Lrp protein binds to multiple sites upstream of the gcv promoter, from about bp -92 to bp -229. The results suggest that the gcv regulatory region is complex, with numerous cis-acting sites that are required for normal gcv expression.
我们构建了一组位于gcv启动子上游的缺失片段，并分析了这些缺失对gcvT-lacZ基因融合表达的影响。一个在转录起始位点（+1）上游-313位处终止的缺失导致gcvT-lacZ表达水平降低，但其融合体仍可被甘氨酸诱导并被嘌呤抑制。一个在-169位处终止的缺失导致gcvT-lacZ融合体丧失了GcvA和Lrp介导的激活作用。δ-313和δ-169的端点也确定了一个使gcvT-lacZ表达下调两到三倍的位点。一个在转录起始位点上游-89位处终止的缺失仍显示出PurR介导的抑制作用，这表明PurR介导的抑制作用并非通过直接干扰GcvA和Lrp介导的调控机制。凝胶迁移率变动分析和DNase I足迹分析表明，Lrp蛋白结合在gcv启动子上游多个位点，从大约-92 bp到-229 bp。结果表明，gcv调控区域很复杂，具有许多正常gcv表达所需的顺式作用位点。
