Abstract
Transfer RNAs (tRNAs) read the genetic code, translating nucleic acid sequence into protein. For tRNASer the anticodon does not specify its aminoacylation. For this reason, mutations in the tRNASer anticodon can result in amino acid substitutions, a process called mistranslation. Previously, we found that tRNASer with a proline anticodon was lethal to cells. However, by incorporating secondary mutations into the tRNA, mistranslation was dampened to a nonlethal level. The goal of this work was to identify second-site substitutions in tRNASer that modulate mistranslation to different levels. Targeted changes to putative identity elements led to total loss of tRNA function or significantly impaired cell growth. However, through genetic selection, we identified 22 substitutions that allow nontoxic mistranslation. These secondary mutations are primarily in single-stranded regions or substitute G:U base pairs for Watson-Crick pairs. Many of the variants are more toxic at low temperature and upon impairing the rapid tRNA decay pathway. We suggest that the majority of the secondary mutations affect the stability of the tRNA in cells. The temperature sensitivity of the tRNAs allows conditional mistranslation. Proteomic analysis demonstrated that tRNASer variants mistranslate to different extents with diminished growth correlating with increased mistranslation. When combined with a secondary mutation, other anticodon substitutions allow serine mistranslation at additional nonserine codons. These mistranslating tRNAs have applications in synthetic biology, by creating "statistical proteins," which may display a wider range of activities or substrate specificities than the homogenous form.
Highlights
MISTRANSLATION occurs when an amino acid that differs from that specified by the “standard” genetic code is incorporated into nascent proteins during translation
We previously demonstrated that SUP17, the gene encoding tRNASer, is lethal when modified with a UGG anticodon and transformed into a wild-type yeast strain (Berg et al 2017)
By showing that the Transfer RNAs (tRNAs) suppress the stress sensitivity of a tti2-L187P mutation, we demonstrated that they mistranslate proline codons, a result confirmed by mass spectrometry
Summary
MISTRANSLATION occurs when an amino acid that differs from that specified by the “standard” genetic code is incorporated into nascent proteins during translation. The final aspect of tRNA regulation is their degradation through either the RTD pathway mentioned above, which degrades hypomodified and unstable tRNAs (Chernyakov et al 2008; Whipple et al 2011), or the nuclear exosome, which monitors tRNA modifications and 39 end maturation (Kadaba et al 2004; Schneider et al 2007; Schmid and Jensen 2008) Because of their toxicity (Berg et al 2017), the applications of mistranslating tRNAs to research and biotechnology requires that their activity be regulated. Using a genetic suppression system that requires a proline codon be mistranslated as serine, we selected mistranslating tRNASerUGG variants with a range of activities after random mutagenesis Many of these had increased toxicity at low temperature and upon inhibiting the RTD pathway, suggesting that they destabilize the tRNA, and enabling temperature-sensitive induction of mistranslation. We demonstrate that in combination with the correct secondary mutation, the anticodon of the tRNASer can be mutated to mistranslate arginine, glutamine, phenylalanine, and ochre stop codons, expanding the mistranslation potential of this tRNA
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