Efficient GFP mutations profoundly affect mRNA transcription and translation rates.

Green fluorescent protein (GFP) variants with higher expression efficiencies have been generated by mutagenesis. Favorable mutations often improve the folding of GFP. However, an effect on protein folding fails to explain the efficiency of several other GFP mutations. In this work, we demonstrate that mutations of the GFP open reading frame and untranslated regions profoundly affect mRNA transcription and translation efficiencies. The removal of the GFP 5' untranslated region halves the transcription rate of the GFP gene, but hugely improves its translation rate. Mutations of the GFP open reading frame or the addition of peptide sequences differentially reduce the GFP mRNA transcription rate, translation efficiency and protein stability. These previously unrecognized effects are demonstrated to be critical to the efficiency of GFP mutants. These findings indicate the feasibility of generating more efficient GFP variants, with optimized mRNA transcription and translation in eukaryotic cells.