Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli

Abstract

In Escherichia coli, the isoleucine codon AUA occurs at a frequency of about 0.4% and is the fifth rarest codon in E. coli mRNA. Since there is a correlation between the frequency of codon usage and the level of its cognate tRNA, translational problems might be expected when the mRNA contains high levels of AUA codons. When a hemagglutinin from the influenza virus, a 304-amino-acid protein with 12 (3.9%) AUA codons and 1 tandem codon, and a mupirocin-resistant isoleucyl tRNA synthetase, a 1,024-amino-acid protein, with 33 (3.2%) AUA codons and 2 tandem codons, were expressed in E. coli, product accumulation was highly variable and dependent to some degree on the growth medium. In rich medium, the flu antigen represented about 16% of total cell protein, whereas in minimal medium, it was only 2 to 3% of total cell protein. In the presence of the cloned ileX, which encodes the cognate tRNA for AUA, however, the antigen was 25 to 30% of total cell protein in cells grown in minimal medium. Alternatively, the isoleucyl tRNA synthetase did not accumulate to detectable levels in cells grown in Luria broth unless the ileX tRNA was coexpressed when it accounted for 7 to 9% of total cell protein. These results indicate that the rare isoleucine AUA codon, like the rare arginine codons AGG and AGA, can interfere with the efficient expression of cloned proteins.In most organisms, there is a noticeable preference for certain codons in highly expressed genes with a subset of so-called rare codons that are generally found in poorly expressed genes (20,22). While this has some interesting evolutionary ramifications for codon selection, there is a more pragmatic problem that these rare codons present with respect to the overexpression of heterologous proteins in a host such as Escherichia coli. There appears to be a correlation between the frequency of codon usage and the level of its cognate tRNA (10). Thus, the expression of mRNA molecules containing a large number of these codons, or tandem rare codons, can result in translational problems. If the level of certain tRNAs was low, then one would predict translational difficulties in decoding mRNA species containing large numbers of these rare codons. This situation would be exacerbated when that particular heterologous mRNA was suddenly induced and represented the major species inside the cell. Since the ribosomes would be more likely to encounter that heterologous mRNA, there would be a need for increased levels of the appropriate acylated rare tRNA species. In the absence of these acylated molecules, the ribosomes would stall while awaiting the appropriate tRNA species (17,18,21,22,31,32), and a stalled ribosome is more likely to produce translational errors such as frameshifts or hops. Such effects have been found when the heterologous mRNA contained the rare codons AGG and AGA (11,18,23,24). This paper addresses the effect of another rare codon, namely, AUA, on the quality and quantity of heterologous proteins expressed in E. coli.In E. coli, the AUA codon is used at a freque...

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