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

Abstract

CCA-adding enzymes are highly specific RNA polymerases that synthesize and maintain the sequence CCA at the tRNA 3'-end. This nucleotide triplet is a prerequisite for tRNAs to be aminoacylated and to participate in protein biosynthesis. During CCA-addition, a set of highly conserved motifs in the catalytic core of these enzymes is responsible for accurate sequential nucleotide incorporation. In the nucleotide binding pocket, three amino acid residues form Watson-Crick-like base pairs to the incoming CTP and ATP. A reorientation of these templating amino acids switches the enzyme's specificity from CTP to ATP recognition. However, the mechanism underlying this essential structural rearrangement is not understood. Here, we show that motif C, whose actual function has not been identified yet, contributes to the switch in nucleotide specificity during polymerization. Biochemical characterization as well as EPR spectroscopy measurements of the human enzyme reveal that mutating the highly conserved amino acid position D139 in this motif interferes with AMP incorporation and affects interdomain movements in the enzyme. We propose a model of action, where motif C forms a flexible spring element modulating the relative orientation of the enzyme's head and body domains to accommodate the growing 3'-end of the tRNA. Furthermore, these conformational transitions initiate the rearranging of the templating amino acids to switch the specificity of the nucleotide binding pocket from CTP to ATP during CCA-synthesis.

Keywords: CCA-adding enzyme; CCA-addition; DEER; EPR spectroscopy; tRNA; tRNA maturation; tRNA nucleotidyltransferase.

Figure 1.

Conserved motifs in the N-terminally located catalytic core of class II tRNA nucleotidyltransferases. (A) The upper bar diagram shows the consensus sequences of motifs A to E in a linear scale model. The individual domains are indicated. The lower part represents an N-terminal zoom-in of the catalytic motifs. The flexible loop element (L) located between motifs A and B is indicated in blue. (B) In the co-crystal structure of a bacterial class II tRNA nucleotidyltransferase (top row: ribbon model, bottom row: surface representation) with a bound tRNA primer (grey) and an ATP analogue (see ref.¹⁰), these motifs are color-coded corresponding to the bar presentation above. It is evident that motif C, indicated in red, has no direct contact to the bound tRNA substrate but is located on the enzyme site opposite to the tRNA binding region and the catalytic core.

Figure 2.

Biochemical analysis of conserved amino acids in motif C of the human CCA-adding enzyme. The conserved amino acid positions D139, G143 and R153 were individually replaced by alanine, and the resulting enzyme variants were tested for CCA-addition using a radioactively labeled human mitochondrial tRNA^Tyr. On the denaturing polyacrylamide gel, the G143A and R153A variants showed the addition of a complete CCA-end comparable to the wt enzyme, while variant D139A exhibits a severely reduced A incorporation. M, mock incubation of tRNA^Tyr without enzyme. The table shows the kinetic parameters for the A-addition on tRNA-CC. While K_M values do not differ significantly between wild-type enzyme (wt) and D139A variant (p = 0.8), the k_cat value of the mutant enzyme form shows a 15-fold reduction compared to the wt (p = 0.003). Correspondingly, the k_cat/K_M values show a similar reduction for the D139A variant. The unaffected K_M values indicate that both wt and D139A enzyme forms have a similar affinity for ATP, showing that this substrate is bound by the mutant enzyme with equal efficiency.

Figure 3.

(A) Crystal structure of the human CCA-adding enzyme with spin label rotamers at positions 87 and 334 (grey stick models calculated using MMM 2013; see ref.²⁵). All motifs are colored according to Figure 1. Residue D139 is highlighted in stick representation (red). The second intrinsic cysteine residue located at position 344 (green) was replaced by alanine prior to spin labeling. In the neck region, α-helices h8 and h11, forming an additional spring element as described by Toh et al., are indicated. (B) The introduction of the spin labels has no effect on the CCA-addition to radioactively labeled human mitochondrial tRNA^Tyr. M, mock incubation of the tRNA in the absence of enzymes. The labeled wt and D139A mutant are indicated by the asterisk. These enzyme preparations show a CCA-adding activity that is indistinguishable from that of the unlabeled versions, with a full CCA end added by the wt* enzyme and a reduced A-addition catalyzed by the D139A* variant.

Figure 4.

(A) DEER experiments and measured interspin distance distributions. Left column: Background corrected form factors F(t) of the averaged DEER traces of HsaCCA H87CR1/C334R1 (black, thin lines) and HsaCCA D139A/H87R1/C334R1 (red, thin lines) for different substrate incubations. Fitted traces for HsaCCA H87CR1/C334R1 (black, bold lines) and HsaCCA D139A/H87R1/C334R1 (red, bold lines) were used to calculate the corresponding interspin distance distributions. Right column: Amplitude-normalized interspin distance distributions for HsaCCA D139A/H87R1/C334R1 (black line) and HsaCCA D139A/H87R1/C334R1 (red line) for different substrate incubations. The area of the distance distribution for apo HsaCCA H87R1/C334R1 (light grey) is displayed as a visual reference for all distance distributions, while the calculated interspin distances for apo HsaCCA H87R1/C334R1 (blue line) are only shown in the first distance distribution. (B) Mechanistic model for different motif C states during CCA-addition. As a spring element, motif C is involved in the adjustment of the catalytic core and the tRNA binding site for the individual reaction steps. In the wt enzyme, motif C (striped box, black) is expanded upon tRNA binding and remains in this conformation for both C-adding steps. In this conformation, the enzyme provides enough space for the growing tRNA. Upon ATP binding, motif C undergoes a slight contraction that might be required for A incorporation and/or limiting the number of added nucleotides. In the D139A enzyme variant (red), motif C (striped box, red) shows no expansion upon tRNA and/or nucleotide binding, even with a growing CC-end on the tRNA substrate. Only in the terminal step, the spring element is significantly extended, leading to an orientation of the tRNA primer end relative to the bound ATP that impedes an incorporation of this nucleotide, resulting in a strongly reduced A-adding activity.

Figure 5.

Tryptophan fluorescence of wild-type CCA-adding enzyme of H. sapiens and the variant carrying the motif C mutation D139A. Upon addition of yeast tRNA^Phe-CC to 1 μM concentration, the fluorescence spectra of both enzyme forms show identical signal quenching (according to student's t-test, none of the values show a significant difference between wt and D139A variant). A further increase in tRNA-CC concentration (3 μM, 6 μM) does not lead to a further signal reduction. As a negative control, BSA was incubated with the corresponding tRNA-CC concentrations. For tRNA concentrations between 0.2 and 1 μM, the fluorescence signal was monitored from 340 to 352 nm. Neither concentration lead to any fluorescence quenching, indicating that the signal reduction of the CCA-adding enzyme variants is not a result of unspecific tRNA binding.

Figure 6.

Proposed structural model derived from the comparison of the differences between HsaCCA and HsaCCA D139A as revealed after a 7.5 ns MD simulation (YASARA). All motifs are colored according to Fig. 1. (A) In HsaCCA, the aspartate residue at position 139 (stick model, red) forms two hydrogen bonds with the backbone Nα positions of glycine 143 and tyrosine 144. This interaction stabilizes the α-helical conformation and contributes to the spring function of motif C. The flexible loop (blue) forms a salt bridge to glutamate 166 (stick representation, orange) located in motif D. (B) In HsaCCA D139A the spring function of motif C is impaired due to the exchange of D139 with alanine (stick representation, red), removing the stabilizing hydrogen bonds. As a consequence, the catalytic head domain rotates from its initial position and the flexible loop (blue) disconnects from E164 (stick model, orange). The changes in the Cα- Cα distances between H87 and C334 are insignificant (HsaCCA: 40.1 Å, HsaCCA D139A: 40.8 Å).