The 3′ Codon Context Effect on UAG Suppressor tRNA is Different in Escherichia coli and Human Cells

We have compared the effect of 3′ context on the efficiency of nonsense suppressor tRNAs in Escherichia coli and human cells. Plasmids containing amber (UAG) termination codons were constructed in the vector pRSVβgal by oligonucleotide insertion at an N-terminal location in a lacZ fusion. A family of identical vectors was prepared with either A, C, G or U as the first 3′ base following the stop codon. These derivatives of pRSV/βgal were expressed in E. coli as stable plasmids, or transiently in human 293 cell tissue culture. Nonsense suppression was monitored using enzyme assays for β-galactosidase. In E. coli the efficiency of a plasmid-borne bacterial tRNAtrp UAG suppressor varied A > G > C = U. When the same lacZ reporter vectors were cotransfected with a human tRNAser UAG suppressor plasmid into human cells, context effects of a different nature were detected. Double reciprocal analysis of close-response experiments were used to show that the efficiency of suppression varied C > G > U = A. The discovery of different codon context effects on nonsense suppression in human cells suggests that the interaction between mammalian tRNAs or release factors and their target codons may have different characteristics from those in bacteria.