Rachel Sonntag

Transfer ribonucleic acid (tRNA) represents one of the most chemically modified RNA species, carrying numerous epigenetic modifications that fine-tune its structure and function. While modifications on the tRNA body maintain and enhance molecular stability, those in the anticodon stem loop are essential for improving translation efficiency and fidelity. This thesis focused on two distinct tRNA modifications in Saccharomyces cerevisiae: cyclic threonylcarbamoyladenosine (ct⁶A) at position 37 of the anticodon stem loop, catalyzed by the heterodimer of threonylcarbamoyl dehydratases Tcd1 and Tcd2, and 2’-O-ribose methylation at position 4 (Nm4) in the amino acid acceptor stem, catalyzed by the tRNA methyltransferase Trm13.  Investigation of these enzymes contributes to our understanding of how tRNA modifications are post-transcriptionally installed and advances the knowledge of epitranscriptomics in genetic regulation and cellular fitness.

The primary goal of this work was to elucidate the reaction mechanisms and structure-function relationships of Tcd1-Tcd2 and Trm13. A multidisciplinary approach combining biochemical characterization, mutagenesis, and activity assays was employed to identify the essential residues and structural features required for their catalytic activity. For Tcd1-Tcd2, our findings indicate that Tcd1 as well as Tcd2 are necessary to produce ct⁶A and that their C-terminal domains of unknown function are required for enzymatic activity and substrate binding. Notably, our results suggest a new catalytic mechanism for the eukaryotic Tcd1-Tcd2 complex, distinct from that of bacterial homologs. Parallel investigations into Trm13 focused on its interaction with the conserved double helix of the acceptor stem. By performing truncation and mutagenesis of the N-terminal tail and its zinc finger (ZnF) motifs in both S. cerevisiae and Schizosaccharomyces pombe homologs, we revealed distinct tRNA-binding strategies. While spTrm13 can bind tRNA without its ZnF motif, scTrm13 requires at least one ZnF for successful interaction. Furthermore, activity assays developed via liquid chromatography–mass spectrometry (LC-MS) demonstrated that 2’-O-ribose methylation occurs independently of other pre-existing modifications on the tRNA body. In summary, this work contributes details of how tRNA modifications are posttranscriptionally installed and advances our understanding of the role of epitranscriptomics in genetic regulation and cellular fitness.

 

Thesis

R. Sonntag (2025), Mechanistic Analysis of Site-Specific tRNA Modifications in Saccharomyces cerevisiae