tRNA nucleotidyltransferases: ancient catalysts with an unusual mechanism of polymerization

Abstract

RNA polymerases are important enzymes involved in the realization of the genetic information encoded in the genome. Thereby, DNA sequences are used as templates to synthesize all types of RNA. Besides these classical polymerases, there exists another group of RNA polymerizing enzymes that do not depend on nucleic acid templates. Among those, tRNA nucleotidyltransferases show remarkable and unique features. These enzymes add the nucleotide triplet C-C-A to the 3'-end of tRNAs at an astonishing fidelity and are described as "CCA-adding enzymes". During this incorporation of exactly three nucleotides, the enzymes have to switch from CTP to ATP specificity. How these tasks are fulfilled by rather simple and small enzymes without the help of a nucleic acid template is a fascinating research area. Surprising results of biochemical and structural studies allow scientists to understand at least some of the mechanistic principles of the unique polymerization mode of these highly unusual enzymes.

Fig. 1

Crystal structures of a class I CCA-adding enzyme (A. fulgidus) in comparison with a class I poly(A) polymerase (S. cerevisiae). The rainbow color indicates the sequential order of the individual enzyme regions, starting from N- (blue) to C-terminus (red). Both enzymes show a catalytic cleft that is formed by the head, neck, and body domains. The CCA-adding enzyme carries an additional tail domain. The structures are extracted from the corresponding pdb entries [29, 90]

Fig. 2

Nucleotide-binding pocket of a class I CCA-adding enzyme with templating region. A highly conserved arginine residue of the enzyme (green) as well as phosphate groups of the tRNA backbone (grey) form specific hydrogen bonds with the bound nucleotides (red). The collaboration of both protein and tRNA is responsible for an efficient and accurate templating during CCA-addition. The shown structures are parts of the pdb entries of the A. fulgidus CCA-adding enzyme [33]

Fig. 3

Polymerization mechanism of CCA-addition according to the general two-metal ion-catalyzed reaction. The two metal ions (Me²⁺, grey balls) are bound to the two catalytically important carboxylates. Metal ion A deprotonates the 3′-OH group of the tRNA primer (grey) and activates the resulting 3′-O⁻ (red) for an attack at the α-phosphate of the incoming nucleotide (red arrows). The second metal ion B stabilizes the triphosphate moiety of the NTP and facilitates the leaving of the pyrophosphate group. Modified from [40]

Fig. 4

Structural organization of class II CCA-adding enzymes. Upper part The catalytically important motifs are indicated as red boxes with consensus sequences in the rainbow bar. In the three-dimensional structure (CCA-adding enzyme from Thermotoga marimita [46]), these motifs are indicated in dark red in the head and neck domain. Lower part The nucleotide-binding site (green) recognizes the incoming CTP and ATP (red) by the formation of Watson/Crick-like hydrogen bonds between the amino acid template EDxxR and the corresponding edge of the bases. The nucleotide-binding site structure is derived from the pdb entry of the B. stearothermophilus CCA-adding enzyme [45]

Fig. 5

Binding of the tRNA primer in class I and II CCA-adding enzymes. For a better visibility of tRNA and bound ATP, the orientation of the enzymes was reversed compared to Figs. 1 and 4. In order to compare the entry sites of the tRNA primer (blue, strand with 3′-OH, green, complementary strand with 5′-end), the bound ATP (red) is oriented in identical positions in the proteins (grey). While in class I enzymes (left), the primer enters the enzyme from the left, it is bound to class II from the top. Due to these dramatically different orientations, the 3′-terminal base of the primer in the class II enzyme has to leave its original position in the tRNA and has to rotate for about 90° in order to be in a position that allows a stacking interaction with the bound NTP and an efficient nucleotide transfer. Structures are extracted from the corresponding pdb entries [32, 33]

Fig. 6

Repair scenario catalyzed by a class II CCA-adding enzyme with phosphatase activity. Due to hydrolytic damage (black bolt), the tRNA (grey) has lost the terminal A residue of the CCA-end and carries now a 2′3′-cyclo phosphate. The phosphatase center of the CCA-adding enzyme removes this phosphate group and converts the tRNA 3′-end into a standard primer, carrying 2′- and 3′-OH groups. Subsequently, the nucleotidyltransferase activity of the CCA-adding enzyme incorporates a new A residue and restores the CCA terminus

Fig. 7

Modular evolution of class II nucleotidyltransferases as supported by experimental and phylogenetic data. From a starting CCA-adding enzyme (center), new enzymes emerge by the insertion/deletion/exchange of protein modules. Individual events of this scenario are presented in a clockwise orientation: An mRNA-binding site module transforms the enzyme into a poly(A) polymerase. The deletion of the lever loop module restricts the activity to CC-addition. Exchange of large N- or C-terminal parts lead either to a poly(CCA) polymerase or to a further CCA-adding enzyme with the catalytic core of poly(A) polymerase. The insertion of the HD domain introduces a phosphatase activity. The evolution of A-adding enzymes, however, is not yet clarified (dashed arrow with question mark). The individual insertions, deletions, and replacements are indicated only at approximate positions

Fig. 8

Evolution of class I and class II CCA-adding enzymes as well as poly(A) polymerases. Starting from a primitive minimal nucleotidyltransferase MNT, class I and class II enzymes emerged rather early by the addition of functional domains. Class I split into two subgroups consisting of archaeal CCA-adding enzymes and eukaryotic poly(A) polymerases. In class II, however, CCA-adding enzymes probably emerged first and evolved then into enzymes with different (poly(A) polymerases) or partial activities (CC-adding enzymes). The evolution of A-adding enzymes is not understood (indicated by the dashed arrows and the question marks). It is possible that these enzymes present the ancestral state, leading to CCA-adding enzymes and poly(A) polymerases. Vice versa, it is also possible that one of these activities is the progenitor of the A-adding enzymes. Modified after [5]. Graphical presentations are based on the available pdb entries of the individual enzymes