Modulating Mistranslation Potential of tRNA Ser in Saccharomyces cerevisiae

• Published in: Genetics (Austin) Vol. 213; no. 3; p. 849
• Main Authors: Berg, Matthew D, Zhu, Yanrui, Genereaux, Julie, Ruiz, Bianca Y, Rodriguez-Mias, Ricard A, Allan, Tyler, Bahcheli, Alexander, Villén, Judit, Brandl, Christopher J
• Format: Journal Article
• Language: English
• Published: United States 01.11.2019
• Subjects: Base Pairing; Loss of Function Mutation; Protein Biosynthesis; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Transfer, Ser - genetics; RNA, Transfer, Ser - metabolism; Saccharomyces cerevisiae; synthetic biology; tRNA modifications; statistical proteins; mistranslation; serine tRNA
• ISSN: 1943-2631

Transfer RNAs (tRNAs) read the genetic code, translating nucleic acid sequence into protein. For tRNA the anticodon does not specify its aminoacylation. For this reason, mutations in the tRNA anticodon can result in amino acid substitutions, a process called mistranslation. Previously, we found that tRNA 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 tRNA 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 tRNA 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.