Evolution of tRNA nucleotidyltransferases: A small deletion generated CC-adding enzymes

Abstract

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

Domain movements during CCA-addition: A new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases

“…To rule out that the reduced A-addition represents an effect specific to the tRNA Tyr substrate, the determination of MichaelisMenten kinetic parameters was conducted with another substrate, yeast tRNA Phe , one of the most commonly used substrates in CCA adding reactions. 16,17,21 To analyze the efficiency of the terminal A-addition. While the observed K M values of 0.09 mM and 0.11 mM did not differ significantly between the wt enzyme and the D139A variant (p D 0.8), the latter variant showed a strongly reduced k cat value of 0.008 s ¡1 , compared to 0.119 s…”

“…16,17 Figure 2. Biochemical analysis of conserved amino acids in motif C of the human CCA-adding enzyme.…”

“…16 Interestingly, this loop is missing in the above mentioned CCadding enzymes, explaining the restricted activity of these nucleotidyltransferases. 17 While the connecting motif C shows a rather low level of sequence conservation with only three predominant positions (Dx (3,4) Gx (9) R), mutations therein can cause a temperaturesensitive phenotype, indicating an important role in catalysis. 18 Yet, crystal structures show that the location of this motif in the enzyme does not support any interaction with tRNA or nucleotides in the catalytic core but helps to correctly position head (with motifs A and B) and neck domains (motif D) for proper catalysis (Fig 1B).…”

Characterization of tRNACys processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control

“…In organelles, the tRNA nucleotidyltransferase only has to add two nucleotides, C and A, to the 39 end because a high number of plastid tRNA genes encode a C at the 39 end (Mayer et al, 2000;Tomita et al, 1996). Aquifex aeolicus, Deinococcus radiodurans, some cyanobacteria and recently Bacillus halodurans, Bacillus clausii and Geobacter sulfurreducens have been found to contain separate enzymes for the addition of A and C residues to tRNA in vivo (Bralley et al, 2005(Bralley et al, , 2009Neuenfeldt et al, 2008;Seth et al, 2002;Tomita & Weiner, 2001. In Streptomyces coelicolor less than a third of the tRNA genes encode the CCA end and the product of SCO3896, the S. coelicolor CCase, is essential in this process (Bralley et al, 2006).…”

“…In some organisms a single CCase modifies the 39 end of damaged or immature tRNAs in a template-independent manner. Interestingly, it has been shown that in Aquifex aeolicus (Tomita & Weiner, 2001), Synechocystis sp., Deinococcus radiodurans (Tomita & Weiner, 2001), Bacillus halodurans (Bralley et al, 2005), Bacillus clausii (Neuenfeldt et al, 2008) and Geobacter sulfurreducens (Bralley et al, 2009) there are two enzymes, one of which adds the CC dinucleotide and one that adds the terminal A. In B. subtilis there is only a single gene coding for CCase (Raynal et al, 1998).…”