Quality control despite mistranslation caused by an ambiguous genetic code

Abstract

A high level of accuracy during protein synthesis is considered essential for life. Aminoacyl-tRNA synthetases (aaRSs) translate the genetic code by ensuring the correct pairing of amino acids with their cognate tRNAs. Because some aaRSs also produce misacylated aminoacyl-tRNA (aa-tRNA) in vivo, we addressed the question of protein quality within the context of missense suppression by Cys-tRNA(Pro), Ser-tRNA(Thr), Glu-tRNA(Gln), and Asp-tRNA(Asn). Suppression of an active-site missense mutation leads to a mixture of inactive mutant protein (from translation with correctly acylated aa-tRNA) and active enzyme indistinguishable from the wild-type protein (from translation with misacylated aa-tRNA). Here, we provide genetic and biochemical evidence that under selective pressure, Escherichia coli not only tolerates the presence of misacylated aa-tRNA, but can even require it for growth. Furthermore, by using mass spectrometry of a reporter protein not subject to selection, we show that E. coli can survive the ambiguous genetic code imposed by misacylated aa-tRNA tolerating up to 10% of mismade protein. The editing function of aaRSs to hydrolyze misacylated aa-tRNA is not essential for survival, and the EF-Tu barrier against misacylated aa-tRNA is not absolute. Rather, E. coli copes with mistranslation by triggering the heat shock response that stimulates nonoptimized polypeptides to achieve a native conformation or to be degraded. In this way, E. coli ensures the presence of sufficient functional protein albeit at a considerable energetic cost.

Fig. 1.

Scheme of missense suppression. Misacylating ProRS forms Pro-tRNA^Pro and Cys-tRNA^Pro (32). When correctly acylated Pro-tRNA^Pro decodes the mRNA of thyA:146_CCA^C→P, the resulting inactive ThyA[146P] does not complement the ΔthyA strain. In contrast, decoding the mRNA of thyA:146_CCA^C→P with misacylated Cys-tRNA^Pro gives a wild-type ThyA[146C] and rescues thymine auxotrophy. In the absence of a misacylating aaRS, the Upper scheme prevails, and suppression is not observed.

Fig. 2.

Missense suppression by misacylated aa-tRNAs. (Top) Cys-tRNA^Pro corrects a nonfunctional thyA gene. The proS genes from G. lamblia (GL), Borrelia burgdorferi (BB), or E. coli (EC) and the thyA:146_CCA^C→P (thyA⁻) allele were expressed in the E. coli ΔthyA strain at 37 °C on minimal medium (MM) A agar plates with or without thymine (20 μg/ml). (Middle) Glu-tRNA^Gln corrects a nonfunctional tryptophan synthase gene. The H. pylori ND-GluRS (HPgluS2) and the mutant trpA:49_CAA^E→Q (trpA⁻) or wild-type (trpA) allele were expressed in the E. coli trpA⁻ strain at 37 °C on minimal medium B agar plates with or without tryptophan (20 μg/ml). (Bottom) Ser-tRNA^Thr corrects a nonfunctional β-lactamase gene. The thrS derivatives and the wild-type (bla) or mutant bla:68_ACA^S→T (bla⁻) allele were expressed in the E. coli thrS^ts strain at 42 °C (nonpermissive temperature) on minimal medium C agar plates with or without ampicillin (20 μg/ml).

Fig. 3.

Proteases are essential for coping with mismade proteins. (Left) Growth curves of protease-replete derivatives. The E. coli strain MG1655 (○) was transformed with the nondiscriminating HPgluS2 (●), the GLproS (□), or the nondiscriminating DRaspS2 (■) and grown at 37 °C in minimal medium. (Right) Growth curves for the protease-deficient strain derivatives. The isogenic strain KY2350 was transformed with the same set of misacylating aaRSs and grown under the same conditions as described above.

Fig. 4.

Asp-tRNA^Asn caused misincorporation in the reporter protein DHFR. The folA:3_AAT^S→N missense mutant allele was expressed in the E. coli trpA:60_AAT^D→N strain containing the nondiscriminating DRaspS2 with (Upper) or without (Lower) tryptophan. Tryptic digests of both DHFR protein preparations were methylated before MALDI-TOF MS analysis. Two N-terminal fragments of interest are shown: MINLIAALAVD*R (monoisotopic mass, MH⁺ = 1,327.78) and MI D*LIAALAV D*R (monoisotopic mass MH⁺ = 1,342.78). The ratio of absolute intensities of peaks at m/z 1,327.78 and 1,342.78 corresponds to the Asp/Asn ratio of the amino acid at position 3.