Characterization of the gcv control region from Escherichia coli

Abstract

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.