Abstract EANA2026-38

The accurate decoding of the genetic code is supported by two molecular processes. One is the assignment of amino acids to tRNAs by aminoacyl-tRNA synthetases, and the other is codon reading by the anticodon of tRNAs on the ribosome. These two processes must have been connected before, or at least by, the stage of the Last Universal Common Ancestor (LUCA). However, how this connection was established remains unclear. In this presentation, we discuss this problem using ancestral molecules related to the translation system.

We have previously reconstructed ancestral sequences of aminoacyl-tRNA synthetases and examined the possible evolution of amino acid specificity, especially by using class IIa aminoacyl-tRNA synthetases as examples (Furukawa et al. 2022). These studies addressed how amino acids may have been assigned to tRNAs in an early stage of translation. However, amino acid assignment by synthetases is only one process of the translation system. To discuss how the genetic code was established, it is also necessary to examine what kinds of tRNAs existed at that stage and how they decoded codons.

We have also studied the evolution of genetic code, mainly based on the atypical genetic codes found in the modern genetic systems such as metazoan mitochondrial system (cf. Watanabe & Yokobori 2011). From this viewpoint, Watanabe and Yokobori (2014) proposed that the early decoding system may have used unmodified tRNAs before the establishment of present RNA modification systems. In that model, U34-tRNAs (34th position of tRNA is the first position of anticodon) decode family-box codons, whereas separation of NNY and NNR codons in two-codon sets may have depended on competition between G34-tRNAs and U34-tRNAs, or C34-tRNAs in some cases. 

To examine this model experimentally we reconstructed tRNA sequences corresponding to LUCA, the Last Archaeal Common Ancestor (LACA), and the Last Bacterial Common Ancestor (LBCA), by ancestral sequence reconstruction. The reconstructed tRNAs were compared with modern tRNAs, especially with respect to identity elements for aminoacylation. Some Commonote tRNAs were synthesized after changing their anticodons, and their functions were tested in an Escherichia coli cell-free translation system using reporter genes containing selected ACN codons.

In this presentation, we mainly discuss ancestral tRNAs, with our previous and recent studies on ancestral aminoacyl-tRNA synthetases. Direct examination of the relationship between ancestral tRNAs and ancestral aminoacyl-tRNA synthetases remains for future work. Here, we discuss what can be learned from reconstructed tRNAs themselves for considering the early translation system.

 

References

Furukawa, R., S. Yokobori, R. Sato, T. Kumagawa, M. Nakagawa, K. Katoh, & A. Yamagishi (2022) Amino acid specificity of ancestral aminoacyl tRNA synthetase prior to the last universal common ancestor Commonote commonote. J. Mol. EvoL. 90(1):73-94 

Watanabe, K. & S. Yokobori (2011) tRNA modification and genetic code variations in animal mitochondria. J. Nucleic Acids. Article ID 623095, doi:10.4061/2011/623095.

Watanabe, K. & S. Yokobori (2014) How the early genetic code was established? -Inference from the analysis of extant animal mitochondrial decoding systems-. Chemical Biology of Nucleic Acids: Fundamentals and Clinical Applications, pp. 25-40, Springer-Verlag Berlin Heidelberg.