The methyltransferase TrmA facilitates tRNA folding through interaction with its RNA-binding domain

Abstract

tRNAs are the most highly modified RNAs in all cells, and formation of 5-methyluridine (m5U) at position 54 in the T arm is a common RNA modification found in all tRNAs. The m5U modification is generated by the methyltransferase TrmA. Here, we test and prove the hypothesis that Escherichia coli TrmA has dual functions, acting both as a methyltransferase and as a tRNA chaperone. We identify two conserved residues, F106 and H125, in the RNA-binding domain of TrmA, which interact with the tRNA elbow and are critical for tRNA binding. Co-culture competition assays reveal that the catalytic activity of TrmA is important for cellular fitness, and that substitutions of F106 or H125 impair cellular fitness. We directly show that TrmA enhances tRNA folding in vitro independent of its catalytic activity. In conclusion, our study suggests that F106 and H125 in the RNA-binding domain of TrmA act as a wedge disrupting tertiary interactions between tRNA's D arm and T arm; this tRNA unfolding is the mechanistic basis for TrmA's tRNA chaperone activity. TrmA is the second tRNA modifying enzyme next to the pseudouridine synthase TruB shown to act as a tRNA chaperone supporting a functional link between RNA modification and folding.

Figure 1.

Structural representation of TrmA and its interaction with tRNA. (A) Structure of TrmA bound to T arm of tRNA (PDB ID: 3BT7). The target uridine 54 (magenta) is flipped into the active site comprising residues C324 (red) and E358 (orange) which are directly participating in catalysis of methylation. Residues G220 (brown) and Q190 (yellow) also contribute to catalysis by binding SAM (G220) and hydrogen-bonding with the flipped-out U54 (Q190). The tRNA elbow region has been predicted to interact with R51 (purple), F106 (blue) and H125 (cyan) (7). (B) Structural Model for the disruption of D arm and T arm tertiary interactions in tRNA by the F106-H125 wedge in the RNA-binding domain of TrmA. To model the initial interaction of TrmA with full-length tRNA, the T arm of the full-length tRNA structure (PDB ID: 4TRA) was aligned with the T arm bound to TrmA (PDB ID: 3BT7, PyMol). For clarity, the T arm bound to TrmA is not displayed. This structural model shows the steric clash between F106 (blue) and H125 (cyan) in the RNA-binding domain with the interface of the D arm (purple) and T arm (light blue) of the tRNA, in particular with nucleotide G18 (D arm, pink) which forms a tertiary interaction with Ψ55 in the T arm. Thus, F106 and H125 act like a wedge disrupting tertiary interactions within the elbow region in tRNA in order to allow the target U54 in the T arm to flip into the active site (Q190, E358, C324, G220). This tRNA unfolding is the mechanistic basis for the tRNA chaperone activity of TrmA.

Figure 2.

tRNA binding by TrmA wild-type and variants. To determine the affinity of TrmA for tRNA in the presence or absence of 50 μM SAM, 10 nM of radioactively labeled tRNA^Phe was incubated with increasing concentrations of TrmA wt or variants. The percentage of bound RNA was determined by nitrocellulose filtration, and the data were fitted to a hyperbolic equation to determine the dissociation constant (K_D). A representative binding curve is shown for each data set, and dissociation constants measured in at least three independent experiments are summarized in Table 1. (A) Binding of tRNA to TrmA wt in the presence (black) or absence (gray) of SAM. (B) tRNA binding by the catalytically inactive TrmA C324A variant in the presence (black) and absence (gray) of SAM. The experiments in C to E show tRNA binding in the presence of 50 μM SAM for the following TrmA variants: (C) TrmA R51A (black) and TrmA R51A C324A (gray). (D) TrmA F106A (black) and TrmA F106A C324A (gray). (E) TrmA H125A (black) and TrmA H125A C324A (gray). For comparison, the same binding curve for TrmA wt in the presence of SAM is shown in all panels.

Figure 3.

5-methyluridine formation by TrmA wt and variants under single-turnover conditions. (A) To quantitatively determine the single-turnover rate for methylation by TrmA wt, time courses were measured in the quench-flow instrument by rapidly mixing tritium-labeled tRNA (final concentration 1 μM) with 2.5 μM (light gray circles), 5 μM (dark gray squares), and 10 μM (black triangles) TrmA wild-type (preincubated with 50 μM cold SAM) before being quenched with HCl. The resulting data was fitted with a single exponential function to determine the apparent rate of methylation which is independent of the TrmA concentration and therefore not rate-limited by binding of TrmA to tRNA. (B) Single-turnover conditions (5 μM TrmA and 50 μM SAM versus 600 nM tRNA) were used for determining methylation activity rates for TrmA wt (black circles) and variants with substitutions in the active site: Q190A (gray squares), G220D (black triangles), and E358Q (gray diamonds). Rates of methylation for the TrmA variants are determined by fitting to a single-exponential function and are summarized in Table 2. (C) The same single-turnover conditions were applied to compare methylation by TrmA wt (black circles) with the catalytically inactive variant C324A (diamonds) and TrmA F106A (black triangles) and F106E (open gray triangles). (D) The same time course for methylation by TrmA wt is compared to the reactions with R51A (gray squares), H125A (black inverted triangle) and H125E (open inverted gray triangle). All time courses were conducted in duplicate with both data sets shown.

Figure 4.

Contribution of tRNA binding and catalytic activity by TruB to bacterial fitness. (A) Bacterial co-culture competition between E. coli wt and E. coli ΔtrmA (open circles), the ΔtrmA strain expressing TrmA wt protein (closed circles), TrmA F106E (open gray triangles), TrmA H125E (open inverted gray triangles) or TrmA C324A (diamonds). (B) Restriction enzyme and PCR-based bacterial co-culture competition assay between E. coli ΔtrmA expressing the catalytic inactive TrmA C324A variant and E. coli ΔtrmA expressing the tRNA binding variants TrmA F106E or H125E.

Figure 5.

tRNA folding by TrmA in vitro observed by aminoacylation efficiency. (A) Unfolded tRNA^Phe was allowed to refold on ice in the presence or absence of TrmA for different folding times. The folded tRNA was then quantified as the percentage of tRNA that was instantaneously aminoacylated upon addition of an excess of [¹⁴C]phenylalanine and phenylalanine-tRNA-synthetase. Both catalytically active TrmA wt and the inactive variant TrmA C324A are promoting tRNA folding. (B) The tRNA folding assay was conducted with the TrmA variants H125E and F106E which are impaired in binding tRNA. As expected, the tRNA folding activity is reduced (H125E) or abolished (F106E).