Translational quality control occurs at several steps, including amino acid selection by aminoacyl-tRNA synthetases (aaRSs). Phenylalanyl-tRNA synthetase (PheRS) misactivates Tyr but is able to correct the mistake using a proofreading activity named editing. Using Escherichia coli PheRS as a model, we show that PheRS editing is the major proofreading step that prevents incorporation of Tyr at Phe codons during translation. In addition, mischarged aa-tRNAs released by PheRS can be resampled via a trans editing pathway that reduces the overall error rate of aminoacyl-tRNA synthesis, providing an additional quality control step prior to translation elongation.
While many aaRSs, such as PheRS, actively edit noncognate amino acids, editing mechanisms are not evolutionarily conserved and their physiological significance remains unclear. To address the connection between aaRSs and mistranslation, we used the evolutionary divergence of tyrosine editing by PheRS as a model system. Certain PheRSs such as those in Mycoplasma species are naturally error-prone and display a low level of specificity consistent with elevated mistranslation of the proteome. Mycoplasma mobile PheRS (MmPheRS) lacks canonical editing activity, relying instead on alternate low stringency quality control pathways. This mechanism of discrimination is inadequate for organisms where translation is usually more accurate. As a result, MmPheRS failed to support E. coli growth. However, minor changes in the defunct editing domain of the MmPheRS are sufficient to restore amino acid specificity and sustain E. coli growth, indicating that translational accuracy is an evolutionarily adaptable trait. These findings indicate a mechanism by which aaRSs facilitate adaptation to changes in cellular physiology by altering the accuracy of translation of specific codons, which may prove advantageous for growth under different environmental conditions.
In addition to its role in cytoplasmic protein synthesis, PheRS is also essential for the proper functioning of organelles such as mitochondria. Structural and functional studies revealed that rearrangement of the RNA-binding and catalytic domains of human mitochondrial PheRS (mtPheRS) between closed and open states are required for completion of the aminoacylation reaction. These results combined with corroborating SAXS experiments indicate that conformational flexibility of the two functional modules in mtPheRS is essential for its phenylalanylation activity, consistent with the modular evolution of the aaRSs.
Defects in mitochondrial translation can lead to a number of mitochondrial diseases like diabetes, deafness, encephalopathy and other myopathies. Although mutations in mitochondria-encoded tRNAs are known to be pathogenic, it has recently become apparent that mutations in nuclear-encoded components of the mitochondrial translation machinery, such as the mtPheRS, could also lead to disease. Non-synonymous single nucleotide polymorphisms (ns-SNPs) occurring in regions distal to mtPheRS catalytic site, affect overall aminoacylation indirectly through refolding specific defects. Pathogenic mutations in mtPheRS associated with infant cardiomyopathy impact function directly by impairing substrate binding and amino acid activation. Our work sheds light on the effects of pathogenic mutations in mtPheRS, which can provide a molecular basis for related mitochondrial diseases.