Gain of function in a tRNA nucleotidyltransferase deletion variant

tRNA nucleotidyltransferases - CCA-adding enzymes - are ubiquitous enzymes catalyzing the template-independent incorporation of the invariant triplet C-C-A to the 3' end of tRNAs. This reaction is essential for the tRNA's functionality. Interestingly, some prokaryotes carry two individual enzymes with partial activities that collaborate in the CCA addition: One enzyme adds the first two C-residues, while the second enzyme incorporates the terminal A-residue. Although these CC- and A-adding enzymes exhibit such restricted activities, they carry the same set of highly conserved motifs in the catalytic core that are required for a complete CCA-addition. In the case of CC-adding enzymes, it could be shown that the deletion of a small flexible loop region between the first two motifs is responsible for the reduced polymerization reaction, as this loop is a prerequisite for switching the enzyme's specificity from CTP to ATP. For the A-adding enzymes, however, the molecular basis of the restricted activity still remains unclear. To identify elements responsible for an exclusive A-incorporating activity, chimeras between A- and CC-adding enzymes of Deinococcus radiodurans were generated. Thereby, the nonconserved C-terminal part of the A-adding enzyme was replaced by the corresponding C-terminus of the CC-adding enzyme. Activity tests revealed that in these chimeras, a full CCA-adding activity was restored. To analyze whether this gain of function is caused by the C-terminus of the CC-adding enzyme, a variant of the A-adding enzyme lacking this part was tested. Surprisingly, this C-terminal deletion variant also showed a complete CCA-adding activity, comparable to that of the chimera. Obviously, the C-terminus of the A-adding enzyme has an inhibitory function and restricts the enzyme's activity to A-incorporation.