Evolution of tRNA nucleotidyltransferases: a small deletion generated CC-adding enzymes.
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 CCA-adding 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 CC-adding 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.
Institute for Biochemistry, University of Leipzig, Brüderstrasse 34, 04103 Leipzig, Germany.
Anne
A
Neuenfeldt
Andrea
A
Just
Heike
H
Betat
Mario
M
Mörl
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D017433:Protein Structure, Secondary; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion
D011993:Recombinant Fusion Proteins; C439999:CC adding enzyme; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase