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. Authors describe for the first time an approach for achieving an increased level of nonsense codon suppression with synthetic suppressor tRNAs in cultured human cells. Authors found that the intracellular concn. of the eukaryotic release factor 1 (eRF1) is a crit. parameter influencing the efficiency of amino acid incorporation by nonsense suppression. Using RNA interference authors were able to lower eRF1 gene expression significantly. Authors 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