Downregulation of eRF1 by RNA interference increases mis-acylated tRNA suppression efficiency in human cells.
The site-specific incorporation of non-natural amino acids into proteins by nonsense suppression has been widely used to investigate protein structure and function. Usually this technique exhibits low incorporation efficiencies of non-natural amino acids into proteins. We describe for the first time an approach for achieving an increased level of nonsense codon suppression with synthetic suppressor tRNAs in cultured human cells. We find that the intracellular concentration of the eukaryotic release factor 1 (eRF1) is a critical parameter influencing the efficiency of amino acid incorporation by nonsense suppression. Using RNA interference we were able to lower eRF1 gene expression significantly. We achieved a five times higher level of amino acid incorporation as compared with non-treated control cells, as demonstrated by enhanced green fluorescent protein (EGFP) fluorescence recovery after importing a mutated reporter mRNA together with an artificial amber suppressor tRNA.
Ilegems
Erwin
E
Institute of Biomolecular Sciences, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland.
Pick
Horst M
HM
Vogel
Horst
H
D000596:Amino Acids / Q000737:chemistry; D001483:Base Sequence; D002460:Cell Line; D003062:Codon; D018389:Codon, Nonsense; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010454:Peptide Termination Factors / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection
D000596:Amino Acids; D003062:Codon; D018389:Codon, Nonsense; D018076:DNA, Complementary; C489002:ETF1 protein, human; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D012343:RNA, Transfer