Perseverance of protein homeostasis despite mistranslation of glycine codons with alanine

By linking amino acids to their codon assignments, transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some human tRNA variants cause amino acid mis-incorporation at a codon or set of codons. We recently found that a naturally occurring tRNA^Ser variant decodes phenylalanine codons with serine and inhibits protein synthesis. Here, we hypothesized that human tRNA variants that misread glycine (Gly) codons with alanine (Ala) will also disrupt protein homeostasis. The A3G mutation occurs naturally in tRNA^Gly variants (tRNA^Gly_CCC, tRNA^Gly_GCC) and creates an alanyl-tRNA synthetase (AlaRS) identity element (G3 : U70). Because AlaRS does not recognize the anticodon, the human tRNA^Ala_AGC G35C (tRNA^Ala_ACC) variant may function similarly to mis-incorporate Ala at Gly codons. The tRNA^Gly and tRNA^Ala variants had no effect on protein synthesis in mammalian cells under normal growth conditions; however, tRNA^Gly_GCC A3G depressed protein synthesis in the context of proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at multiple Gly codons caused by the tRNA^Gly_GCC A3G and tRNA^Ala_AGC G35C mutants, and in some cases, we observed multiple mistranslation events in the same peptide. The data reveal mistranslation of Ala at Gly codons and defects in protein homeostasis generated by natural human tRNA variants that are tolerated under normal conditions. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.