Dosage effect of minor arginyl- and isoleucyl-tRNAs on protein synthesis in an Escherichia coli in vitro coupled transcription/translation system

In Escherichia coli mRNA, the arginine codons AGA/AGG and the isoleucine co don AUA are rarely used with frequencies of about 0.35% and 0.41%, respecti vely. Six genes with a different number of these codons were expressed in a n E. coli in vitro coupled transcription/translation system, which containe d either tRNA prepared from E. coli cells carrying a plasmid with argU and ileX genes encoding rare tRNAs (tRNA(arg) AGA/AGG and tRNA(AUA)(ile)), desi gnated codon-plus tRNA, or normal tRNA from cells lacking the plasmid. Gene s having a low number of the rare codons, such as genes encoding chloramphe nicol acetyltransferase and anti-gp120 single-chain Fv (artificially constr ucted to remove rare codons), were expressed at similar levels using with b oth tRNA preparations. On the other hand, the use of codon-plus tRNA increa sed the expression levels of genes having a relatively large number of the rare codons, including genes encoding archaeal proteins, green fluorescent protein of jelly fish origin, and a single-chain Fv of mouse origin, by abo ut 20% higher than that using normal tRNA.