Anticodon sequence determines the impact of mistranslating tRNA ^Ala variants

Abstract

Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic message is incorporated into proteins and has applications in biotechnology, therapeutics and is relevant to disease. Since the alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in tRNA Ala and the anticodon plays no role in charging, tRNA Ala variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNA Ala anticodon variants on the growth of Saccharomyces cerevisiae . Overall, 36 tRNA Ala anticodon variants decreased growth in single- or multi-copy. Mass spectrometry analysis of the cellular proteome revealed that 52 of 57 anticodon variants, not decoding alanine or stop codons, induced mistranslation when on single-copy plasmids. Variants with G/C-rich anticodons resulted in larger growth deficits than A/U-rich variants. In most instances, synonymous anticodon variants impact growth differently, with anticodons containing U at base 34 being the least impactful. For anticodons generating the same amino acid substitution, reduced growth generally correlated with the abundance of detected mistranslation events. Differences in decoding specificity, even between synonymous anticodons, resulted in each tRNA Ala variant mistranslating unique sets of peptides and proteins. We suggest that these differences in decoding specificity are also important in determining the impact of tRNA Ala anticodon variants.

Mechanisms and Delivery of tRNA Therapeutics

“…Indeed, we noted a correlation between the number of codons read and growth defect just for tRNAs for Gly and Arg. Similar observations were found in a library of tRNA Ala variants in yeast . Rather, we found that the nature of the amino acid replacement according to amino acid similarity and the strength of the anticodon–codon interaction were the dominant factors that contribute to the impact of missense suppressor tRNAs on cell growth …”

“…Similar observations were found in a library of tRNA Ala variants in yeast. 206 Rather, we found that the nature of the amino acid replacement according to amino acid similarity and the strength of the anticodon−codon interaction were the dominant factors that contribute to the impact of missense suppressor tRNAs on cell growth. 45 As noted above, mistranslation does not always lead to detrimental changes in global protein homeostasis.…”

“…Nonsynonymous anticodon variants of tRNA Ala , , tRNA Ser , and tRNA Leu generate missense suppression through amino acid misincorporation, because their cognate aaRSs do not recognize the anticodon. Ser is encoded by six codons, which makes seryl-tRNA synthetase (SerRS) especially flexible in recognizing tRNA Ser with altered anticodons .…”