Efficient GFP mutations profoundly affect mRNA transcription and translation rates

Green fluorescent protein (GFP) variants with higher expression efficiencie s 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 CPP mutations. In this work, we demonstrate th at mutations of the GFP open reading frame and untranslated regions profoun dly affect mRNA transcription and translation efficiencies. The removal of the GFP 5' untranslated region halves the transcription rate of the GFP gen e, but hugely improves its translation rate. Mutations of the GFP open read ing frame or the addition of peptide sequences differentially reduce the GF P mRNA transcription rate, translation efficiency and protein stability. Th ese previously unrecognized effects are demonstrated to he critical to the efficiency of GFP mutants. These findings indicate the feasibility of gener ating more efficient CFP variants, with optimized mRNA transcription and tr anslation in eukaryotic cells. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.