An alternative adaptation strategy of the CCA-adding enzyme to accept noncanonical tRNA substrates in Ascaris suum

Abstract

Playing a central role in translation, tRNAs act as an essential adapter linking the correct amino acid to the corresponding mRNA codon in translation. Due to this function, all tRNAs exhibit a typical secondary and tertiary structure to be recognized by the tRNA maturation enzymes as well as many components of the translation machinery. Yet, there is growing evidence for structurally deviating tRNAs in metazoan mitochondria, requiring a coevolution and adaptation of these enzymes to the unusual structures of their substrates. Here, it is shown that the CCA-adding enzyme of Ascaris suum carries such a specific adaptation in form of a C-terminal extension. The corresponding enzymes of other nematodes also carry such extensions, and many of them have an additional adaptation in a small region of their N-terminal catalytic core. Thus, the presented data indicate that these enzymes evolved two distinct strategies to tolerate noncanonical tRNAs as substrates for CCA incorporation. The identified C-terminal extension represents a surprising case of convergent evolution in tRNA substrate adaptation, as the nematode mitochondrial translation factor EF-Tu1 carries a similar extension that is essential for efficient binding to such structurally deviating tRNAs.

Keywords: CCA-adding enzyme; coevolution; convergent evolution; miniaturized tRNAs; noncanonical tRNAs; tRNA nucleotidyltransferase.

Figure 1

Canonical and noncanonical tRNAs.A, tRNA^Phe from Saccharomyces cerevisiae represents a cloverleaf structure typical for canonical tRNA molecules and has a length of 76 nucleotides. The individual parts of this secondary structure are indicated. B, one of the most extremely truncated noncanonical tRNA is the mitochondrial tRNA^Ile from the nematode Romanomermis culicivorax. Acceptor and anticodon arms show an untypical length, D- and TψC-arms are replaced by unstructured connector elements, reducing the size of the tRNA down to 50 nucleotides. In both tRNAs, the post-transcriptionally added CCA-end is indicated in red.

Figure 2

Sequence alignment of CCA-adding enzymes from vertebrate and nematode representatives. Enzyme sequences from Danio rerio (DreCCA), Bos taurus (BtaCCA), Mus musculus (MmuCCA), Homo sapiens (HsaCCA), Caenorhabditis elegans (CelCCA), Caenorhabditis briggsae (CbrCCA), Romanomermis culicivorax (RcuCCA), and Ascaris suum (AsuCCA) are shown. Identical residues are highlighted in blue, basic and acidic residues in the β-turn element and the C-terminal extension (black bars) are indicated in green and red, respectively. Catalytic core elements (red bars) are highly conserved in all shown CCA-adding enzyme sequences. Besides the five conserved motifs A to E, a flexible loop (involved in domain movement during CCA addition) and a basic/acidic motif (B/A, involved in proof reading of the incorporated nucleotides) are indicated. Only the nematode enzymes carry a C-terminal extension enriched in basic residues, with the longest one (40 positions) in AsuCCA. In the β-turn, AsuCCA is lacking one basic residue in the KR motif of the nematode sequences (green bar). The black arrowhead indicates the position for enzyme fusions and the C-terminal deletion variant. For a correct alignment, the variable N termini containing mitochondrial target sequences were omitted (number of positions indicated in brackets) (24).

Figure 3

Impact of the C-terminal extension on CCA-adding activity and tRNA substrate binding of AsuCCA.A, CCA addition on a canonical (Sce tRNA^Phe from Saccharomyces cerevisiae) and a noncanonical armless tRNA (tRNA^Ile from Romanomermis culicivorax mitochondria). The wt enzyme AsuCCA efficiently adds a complete CCA-end to both tRNA substrates at one or five arbitrary units, respectively (upper gel panels), whereas a version lacking the C-terminal 40 amino acids long extension (AsuCCA ΔC40) is only fully active on the canonical tRNA but adds just one to two C-residues on the armless tRNA, even at increased enzyme concentrations (25 and 50 U) (lower panels). On the noncanonical tRNA, a higher concentration of the wt enzyme is required for CCA addition, a fact that was also described for the corresponding enzyme of R. culicivorax (24). A quantitative comparison of these enzymatic activities and those of the following figures is shown in Fig. S1. B, quantitation of tRNA substrate interaction of AsuCCA wt (blue) and C-terminal deletion variant (pink). In the gel shift analysis, up to 4 μM of the recombinant protein were offered. Both enzyme versions bind efficiently to the canonical tRNA, resulting in dissociation constants of 0.5 to 1.7 μM (left), whereas only the wt enzyme shows high affinity for the armless substrate (K_D = 1.1 μM, right). In contrast, AsuCCA ΔC40 shows a dramatically reduced interaction with this tRNA that does not allow the determination of a dissociation constant (right). The observed supershifts at the highest concentration of AsuCCA are probably the result of protein dimer formation, a reaction that is frequently observed for CCA-adding enzymes at high concentrations in vitro (27, 36, 54). K_D values were determined by nonlinear regression and Hill slope fit. Data are means ± SD; n = 3.

Figure 4

The human CCA-adding enzyme carrying the C-terminal extension of AsuCCA accepts an armless tRNA as substrate.A, when fused to the C-terminal extension of AsuCCA, the human enzyme readily accepts canonical (left) as well as structurally deviating tRNA substrates (right) for efficient CCA-addition. In contrast, the human wt enzyme (lower gel panels) adds only two C-residues to the armless tRNA but not a complete CCA-end (right). On the canonical tRNA, it adds complete CCA-ends, although at a somewhat lower efficiency than the chimera (left). In the bar representation of the enzymes, catalytic core motifs are shown in green, the fusion position of the Ascaris suum C-terminal extension (red) is indicated by the black arrow. B, as described in the literature, the human wt enzyme has a very low affinity to tRNA substrates in general, so that no binding parameters can be determined (black). The C-terminal extension (blue), however, conveys a dramatically increased affinity to both canonical (K_D = 0.7 μM) as well as armless tRNA (K_D = 0.6 μM). Data for wt HsaCCA are taken from Hennig and Philipp (24).

Figure 5

The β-turn of AsuCCA does not convey an efficient CCA addition to a noncanonical tRNA.A, when the β-turn of AsuCCA (orange/red) is replaced by the corresponding element of HsaCCA (green), the resulting chimera AsuCCA-Hsaβ exhibits an unaltered activity on both canonical (left) and noncanonical tRNA substrate (right), leading to complete CCA addition with only one arbitrary enzyme unit. This result shows that the extension (red), which is still present in the chimera, is essential for the recognition of the structurally deviating tRNA. B, in the reciprocal chimera HsaCCA-Asuβ, the AsuCCA β-turn has almost no effect on the activity of HsaCCA. The enzyme is fully active on the cloverleaf tRNA (1 arbitrary unit is sufficient for complete CCA synthesis, left). On the armless substrate, the chimera adds two C-residues, and only at higher enzyme concentrations, a very weak A-incorporation is observed (right). C, reciprocal β-turn replacements do not affect substrate-binding behavior. K_D values could only be determined for AsuCCA-Hsaβ (cyan), as this chimera still carries the C-terminal extension that conveys efficient tRNA binding.

Figure 6

In RcuCCA, the short C-terminal extension makes only a minor contribution to CCA addition to a noncanonical tRNA.A, on the canonical tRNA^Phe, RcuCCA lacking the C-terminal extension of 14 amino acids (RcuCCA ΔC14) is similarly active as the wt enzyme (RcuCCA). On the armless mt-tRNA^Ile, the activity of this deletion variant is somewhat reduced but still leads to complete CCA addition, especially at elevated enzyme concentrations of 25 and 50 U. B, in contrast to the polymerization activity, substrate binding is strongly reduced in RcuCCA ΔC14 (black curves), so that no dissociation constants could be determined. The wt enzyme (blue curves), however, binds both canonical as well as armless tRNA substrates at a K_D of 1.6 and 1.4 μM, respectively. mt-tRNA, mitochondrial tRNA.

Figure 7

Reciprocal exchange of C-terminal extensions between AsuCCA and RcuCCA.A, when introduced into RcuCCA, the C40 extension of AsuCCA does not affect catalysis or substrate interaction, supporting the observation that in this enzyme the β-turn element is the major adaptation to noncanonical tRNA substrates. B, for AsuCCA, replacing the C40 extension with the C14 region of RcuCCA has no effect on CCA addition to the standard tRNA substrate, whereas CCA incorporation to the armless tRNA is reduced and only visible at high enzyme concentrations (25 and 50 U). However, the affinity of the enzyme chimera to both substrates is not affected. This result suggests that the C14 region of RcuCCA is involved in efficient substrate binding but makes a smaller contribution to CCA addition on the armless tRNA compared with the C40 region of AsuCCA.

Figure 8

Evolutionary scenario: The CCA-adding enzymes of nematodes follow two different adaptation strategies to accept noncanonical tRNA substrates. Since truncated tRNAs are an acquired trait (16), the original CCA-adding enzyme probably recognized only canonical and the widely distributed unique D-armless tRNA^Ser. In the CCA-adding enzyme of Ascaris suum, a C-terminal extension evolved as a prerequisite for the acceptance of other structurally deviating tRNAs lacking D-, T-, or even both arms (large red arrowhead). In contrast, the β-turn showed almost no adaptation as it carries only a single basic residue (BN; B = any basic residue; N = any amino acid except basic arginine or lysine; small red arrowhead). This element therefore has its original function in positioning the 3′-end of the tRNA in the catalytic core. In the corresponding enzyme of Romanomermis culicivorax, the main adaptation to noncanonical tRNAs is represented by the two basic positions in the β-turn (BB, large red arrowhead). The small C-terminal extension in this enzyme contributes to the adaptation but to a lesser extent (intermediate red arrowhead).