**2. Material and methods**

Most of the research was done in two databases which include primary sequences and graphical representations of tRNA 2D structures, tRNAdb (http://trna.bioinf.uni-leipzig.de/ DataOutput/) contained more than 12,000 *trn* genes from 579 species belonging to prokaryotes and eukaryotes, whereas in mitotRNAdb (http://mttrna.bioinf.uni-leipzig.de/mtDataOutput/), 30,525 metazoan mt-*trn* genes belonging to 1418 species were recorded [22]. Despite a bias for metazoa, these two databases provide powerful and fast search engines. Alignments were generated by Clustal W (www.ebi.ac.uk/clustalw/), whereas secondary structures were predicted by tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/) [23]. BLAST analyzes were conducted using the website: https://blast.ncbi.nlm.nih.gov/Blast.cgi.

analysis, TAR10 always occurs at high frequencies, whether in prokaryotic, nuclear, or organelle genomes. Values range from 41.1% for fungi to 81.6% for pseudocoelomates. In all the taxa and all tRNA species combined, the percentage of TAG10 triplets is always significantly higher than those of TAA10. The differences are very important in prokaryotic and nuclear genomes, since the percentage of TAA10 is always less than 1, while that of TAG10 is at least 40%. Within the organelle genomes, the difference is smaller but can vary by a factor of 2.5–22. As the TAR10 triplet (principally TAG) is present in at least 40% of the *trn* genes for all taxa and genomic systems combined, it could have been present in *trn* genes of the Last Unicellular Common Ancestor (LUCA), which presumably lived some 3.5–3.8 billion years ago [24]. It is probably an ancestral character which was present in proto-*trn* sequences. As the percentage of TAA10 strongly increases in *trn* genes of organelles, one can ask whether this character was not already present in their bacterial ancestor. It is now assumed that despite their diversity, all mitochondria derive from an endosymbiotic α-proteobacterium which has been integrated into a host cell related to Asgard Archaea approximately 1.5–2 billion years ago [25]. However, the earliest fossils possessing features typical of fungi date to 2.4 billion years ago [26]. Moreover, the eukaryotic cells would be chimeras constituted of an archaebacterium and one or more Eubacteria [27]. In addition, all current models for the origin of eukaryotes suggest that the eukaryotic common ancestor had mitochondria. Therefore, as the level of TAA10 is very low in *trn* genes of α-proteobacteria, it could therefore be a derived trait that may be related to the increase in AT% in mtDNA and/or recognition constraints by mt-aaRSs and modification enzymes. Similarly, it is generally accepted that all chloroplasts and their derivatives are derived from a single cyanobacterial ancestor [28], and in current cyanobacteria, the respective percentage of TAG10 and TAA10 triplets are 62.5 and

**Table 1.** Percentage of TAR10 and ATR49 triplets in various taxa independently of the V-R size.

Metazoan taxa are in italics. *Abbreviation*: Nb, number of *trn* genes. TAR + ATR for % of *trn* genes bearing the two types

True Mitochondrial tRNA Punctuation and Initiation Using Overlapping Stop and Start Codons…

http://dx.doi.org/10.5772/intechopen.75555

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of triplet. \* Mitochondria.

3.6, respectively. The increase in the percentage of TAA10 characterizes organelles.

In all the taxa for all tRNA species combined, the ATR49 triplets are always present in smaller numbers than TAR10. Moreover, their numbers are negligible except in organelle genomes,
