H-NS silences gfp, the green fluorescent protein gene: gfpTCD is a genetically Remastered gfp gene with reduced susceptibility to H-NS-mediated transcription silencing and with enhanced translation

Abstract

The bacterial nucleoid-associated protein H-NS, which preferentially targets and silences AT-rich genes, binds the ubiquitous reporter gene gfp and dramatically reduces local transcription. We have redesigned gfp to reduce H-NS-mediated transcription silencing and simultaneously improve translation in vivo without altering the amino acid sequence of the GFP protein. The green fluorescent protein (GFP) from the jellyfish Ae-quorea victoria is widely used as a reporter of transcriptional activity and protein localization in both eukaryotes and pro-karyotes (reviewed in reference 16). The DNA encoding gfp and many of its variants has a high AT content (60%) and strong predicted DNA curvature, both of which are key fea-tures of DNA that is targeted by the histone-like nucleoid structuring protein (H-NS) (10, 12, 14, 15, 22). H-NS is a global gene silencer that represses transcription by condensing chro-mosome and plasmid DNA in many Gram-negative bacteria. For example, H-NS binds and represses 15 % of the Esche

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