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.
Laboratory of Experimental Oncology and Biotech Group, Department of Cell Biology and Oncology, Instituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy.
A
A
Sacchetti
T
T
El Sewedy
A F
AF
Nasr
S
S
Alberti
D020121:5' Untranslated Regions / Q000235:genetics; D002478:Cells, Cultured; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D017354:Point Mutation; D014176:Protein Biosynthesis; D017510:Protein Folding; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D020121:5' Untranslated Regions; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins