The dnaY gene in E. coli was originally identified in a screen for mutants defective in DNA replication. It has since been shown (Garcia et al., Cell 45:453-9, 1986) that this gene encodes the cognate tRNA for the AGA and AGG arginine codons. It was therefore renamed ArgU. The ArgU tRNA is present at very low levels, and is in fact one of the rarest tRNA’s in E. coli. Brinkmann et al. (Gene 85:109-14, 1989) found that attempts to express heterologous genes containing AGA or AGG codons led to plasmid instability and decreased cell viability. Spanjaard et al. (Nucleic Acids Res. 18:5031-6, 1990) demonstrated that tandem AGA or AGG arginine codons caused frameshifts in the expressed protein when present in an mRNA. This "toxicity", and these frameshifts, could be alleviated by the overexpression of the ArgU gene.
MuDR, the autonomous element of the Mutator family of transposable elements has proven extremely refractory to manipulation in E. coli (Gutierrez et al., Genetics 149:329-346, 1998). Examination of the murA gene, the presumptive transposase, revealed a preponderance of AGA and AGG arginine codons. The murA mRNA has more than 40 AGA or AGG codons, including a triplet AGG and 4 doublets (Fig. 1). It also has a doublet of the cognate codon for the rare IleX tRNA. Overexpression of the ArgU tRNA allows the stable maintenance of murA clones in E. coli, but it does not allow high-level expression of the gene product. Preliminary mutagenesis experiments to alter the AGA and AGG codons to codons more commonly used in E. coli have been successful (Fig. 2).
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