Abstract
Synthesis of the CCA end of essential tRNAs is performed either by CCA-adding enzymes or as a collaboration between enzymes restricted to CC- and A-incorporation. While the occurrence of such tRNA nucleotidyltransferases with partial activities seemed to be restricted to Bacteria, the first example of such split CCA-adding activities was reported in Schizosaccharomyces pombe. Here, we demonstrate that the choanoflagellate Salpingoeca rosetta also carries CC- and A-adding enzymes. However, these enzymes have distinct evolutionary origins. Furthermore, the restricted activity of the eukaryotic CC-adding enzymes has evolved in a different way compared to their bacterial counterparts. Yet, the molecular basis is very similar, as highly conserved positions within a catalytically important flexible loop region are missing in the CC-adding enzymes. For both the CC-adding enzymes from S. rosetta as well as S. pombe, introduction of the loop elements from closely related enzymes with full activity was able to restore CCA-addition, corroborating the significance of this loop in the evolution of bacterial as well as eukaryotic tRNA nucleotidyltransferases. Our data demonstrate that partial CC- and A-adding activities in Bacteria and Eukaryotes are based on the same mechanistic principles but, surprisingly, originate from different evolutionary events.
Highlights
In all domains of life, tRNAs play a crucial role during translation
While the occurrence of such tRNA nucleotidyltransferases with partial activities seemed to be restricted to Bacteria, the first example of such split CCA-adding activities was reported in Schizosaccharomyces pombe
Phylogenetic analyses by us and others indicated that the occurrence of multiple sequences of candidate tRNA nucleotidyltransferases might be more common among genomes of major eukaryotic taxa—i.e., Holozoa, Fungi, Algae, Amoebozoa, and Plants—than previously anticipated [8,35] (Figure 1B)
Summary
In all domains of life, tRNAs play a crucial role during translation. At their 3 terminus, these molecules carry the invariant sequence cytidine–cytidine–adenosine (CCA), which is important for the attachment of the corresponding amino acid and for proper participation of the tRNA in translation [1,2,3]. In all Eukaryotes, most Archaea, and many Bacteria, the CCA triplet is not encoded in the tRNA genes [4,5,6,7,8] and has to be added post-transcriptionally by ATP(CTP):tRNA nucleotidyltransferases (CCA-adding enzymes). For an increasing number of bacteria, it is reported that CCA-addition is performed by the collaborative action of enzymes with partial activities [22,28,29,30,31,32] While both types of enzymes share the five core motifs with class II CCA-adding enzymes, the flexible loop region is absent in CC-adding enzymes, inhibiting the switch from CTP to ATP specificity. PPhhyyllooggeenneettiicc ddiissttrriibbuuttiioonn ooff eeuukkaarryyoottiicc ssppeecciieess bbeeaarriinngg ggeennoommeess wwiitthh mmoorree tthhaann oonnee ttRRNNAA nnuucclleeoottiiddyyllttrraannssffeerraassee ggeennee.. Minduilctiaftuerucantcioerntsa,inqtuyeasbtioountmthaerkpsh,yalongdendeottitcedpobsritainocnh. es indicate uncertainty about the phylogenetic position
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