CCA-adding enzymes are specialized polymerases that add a specific sequence (C-C-A) to tRNA 3 ends without requiring a nucleic acid template. In some organisms, CCA synthesis is accomplished by the collaboration of evolutionary closely related enzymes with partial activities (CC and A addition). These enzymes carry all known motifs of the catalytic core found in CCA-adding enzymes. Therefore, it is a mystery why these polymerases are restricted in their activity and do not synthesize a complete CCA terminus. Here, a region located outside of the conserved motifs was identified that is missing in CC-adding enzymes. When recombinantly introduced from a CCA-adding enzyme, the region restores full CCAadding activity in the resulting chimera. Correspondingly, deleting the region in a CCA-adding enzyme abolishes the A-incorporating activity, also leading to CC addition. The presence of the deletion was used to predict the CC-adding activity of putative bacterial tRNA nucleotidyltransferases. Indeed, two such enzymes were experimentally identified as CC-adding enzymes, indicating that the existence of the deletion is a hallmark for this activity. Furthermore, phylogenetic analysis of identified and putative CCadding enzymes indicates that this type of tRNA nucleotidyltransferases emerged several times during evolution. Obviously, these enzymes descend from CCA-adding enzymes, where the occurrence of the deletion led to the restricted activity of CC addition. A-adding enzymes, however, seem to represent a monophyletic group that might also be ancestral to CCA-adding enzymes. Yet, experimental data indicate that it is possible that A-adding activities also evolved from CCA-adding enzymes by the occurrence of individual point mutations.tRNA maturation ͉ CCA-adding enzyme ͉ flexible loop

In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This raises the question of molecular adaptation of these proteins to their peculiar partner RNAs. The crystal structure of the homodimeric bacterial-type human mitochondrial aspartyl-tRNA synthetase (DRS) confirmed a 3D architecture close to that of Escherichia coli DRS. However, the mitochondrial enzyme distinguishes by an enlarged catalytic groove, a more electropositive surface potential and an alternate interaction network at the subunits interface. It also presented a thermal stability reduced by as much as 12°C. Isothermal titration calorimetry analyses revealed that the affinity of the mitochondrial enzyme for cognate and non-cognate tRNAs is one order of magnitude higher, but with different enthalpy and entropy contributions. They further indicated that both enzymes bind an adenylate analog by a cooperative allosteric mechanism with different thermodynamic contributions. The larger flexibility of the mitochondrial synthetase with respect to the bacterial enzyme, in combination with a preserved architecture, may represent an evolutionary process, allowing nuclear-encoded proteins to cooperate with degenerated organelle RNAs.

For efficient aminoacylation, tRNAs carry the conserved 3′-terminal sequence C-C-A, which is synthesized by highly specific tRNA nucleotidyltransferases (CCA-adding enzymes). In several prokaryotes, this function is accomplished by separate enzymes for CC- and A-addition. As A-adding enzymes carry an N-terminal catalytic core identical to that of CCA-adding enzymes, it is unclear why their activity is restricted. Here, it is shown that C-terminal deletion variants of A-adding enzymes acquire full and precise CCA-incorporating activity. The deleted region seems to be responsible for tRNA primer selection, restricting the enzyme’s specificity to tRNAs ending with CC. The data suggest that A-adding enzymes carry an intrinsic CCA-adding activity that can be reactivated by the introduction of deletions in the C-terminal domain. Furthermore, a unique subtype of CCA-adding enzymes could be identified that evolved out of A-adding enzymes, suggesting that mutations and deletions in nucleotidyltransferases can lead to altered and even more complex activities, as a simple A-incorporation is converted into sequence-specific addition of C and A residues. Such activity-modifying events may have had an important role in the evolution of tRNA nucleotidyltransferases.