# Deciphering the influence of the [4Fe-4S] cluster of tRNA thiolation enzymes on tRNA binding

Sylvain Gervason, Sambuddha Sen, Jean-Luc Ravanat, Sylvain Caillat, Djemel Hamdane, Béatrice Golinelli-Pimpaneau

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Sylvain Gervason, Sambuddha Sen, Jean-Luc Ravanat, Sylvain Caillat, Djemel Hamdane, et al. Deciphering the influence of the [4Fe-4S] cluster of tRNA thiolation enzymes on tRNA binding. RNA, 2025, 31 (6), pp.735-742. 10.1261/rna.080292.124. hal-05283762

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# Deciphering the influence of the [4Fe-4S] cluster of tRNA thiolation enzymes on tRNA binding

Sylvain Gervason¹#, Sambuddha Sen¹#, Jean-Luc Ravanat², Sylvain Caillat², Djemel Hamdane¹*§, Béatrice Golinelli-Pimpaneau¹*

¹Laboratoire de Chimie des Processus Biologiques, Collège de France, CNRS UMR 8829, Sorbonne Université, Paris cedex 05, France.

²University of Grenoble Alpes, CEA, CNRS, Grenoble INP, IRIG, SyMMES, F-38000 Grenoble, France

# same contribution

§ present address: DEV2A, Développement, Vieillissement and Adaptation, CNRS UMR8263, Inserm U1345, Sorbonne Université, Institut de Biologie Paris Seine, Paris 75252, France

*Address correspondence to djemel.hamdane@sorbonne-universite.fr and beatrice.golinelli@college-de-france.fr

## Abstract

Iron-sulfur clusters [Fe-S] play crucial roles in diverse biological reactions, often serving as prosthetic groups for enzymes. Specifically, certain tRNA-modifying enzymes utilize these clusters to catalyze the thiolation of specific nucleosides. While the participation of [4Fe-4S] clusters in such catalytic processes is known, their potential influence on tRNA binding remains unexplored. In this study, we examine the impact of the cluster on the affinity for tRNA of Ttul from the archeon *Methanococcus maripaludis*, an enzyme responsible for the formation of 4-thiouridine at position 8 in tRNAs of archaea and bacteria, as well as *Escherichia coli* TtcA that catalyzes the biosynthesis of 2-thiocytidine at position 32 in bacterial tRNAs. For this purpose, we compare the change of fluorescence properties of judiciously located tryptophans upon tRNA binding between the apo-enzyme (lacking the cluster) and the holo-enzyme (incorporating a reconstituted cluster). Our results indicate that the presence of the [4Fe-4S] cluster does not alter the affinity of the thiolases for tRNA, thus ruling out any direct involvement of the cluster in tRNA binding and emphasizing the purely catalytic role of the [4Fe-4S] cluster in tRNA thiolation.
Introduction

[Fe-S] clusters stand as the oldest metallocofactors on earth (Camprubi et al. 2017), playing key roles in a diverse array of cellular processes. They are essential participants in electron transfer reactions (Yakovlev et al. 2007), contribute to redox and non-redox catalysis (Beinert et al. 1996) and to regulatory mechanisms by acting as sensors (Mettert and Kiley 2015; Crack and Le Brun 2018). While the [2Fe-2S] and [4Fe-4S] states represent the most common forms of [Fe-S] clusters (Beinert 2000), more complex arrangements, with additional heavy metal ions, are present in enzymes such as carbon monoxide dehydrogenases and nitrogenases (Dinis et al. 2016; Jeoung and Dobbek 2018). The irons within the cluster are most often coordinated by the thiolate of cysteine residues of the protein or nitrogen atoms of histidine/arginine residues but occasionally by other ligands, such as the oxygen atoms of aspartate residues (Bak and Elliott 2014; Zhou et al. 2021). In some enzymes, the [4Fe-4S] cluster is coordinated by three protein residues, whereas the fourth iron atom is bound to an external ligand, such as the S-adenosylmethionine (SAM) cofactor in SAM-radical enzymes (Broderick et al. 2023), or a water molecule in aconitase (Castro et al. 2019). This diversity in coordination underscores the adaptability of [Fe-S] clusters, which allows them to ensure a wide range of chemical functions.

A variety of enzymes involved in the posttranscriptional modification of tRNAs, which is crucial for the efficiency and fidelity of genetic translation, rely on [4Fe-4S] clusters (Kimura and Suzuki 2015). They catalyze a large diversity of reactions, encompassing methylations (m²A37 by RlmN), acetylations (cm⁵U34 by Elp3), the formation of an imidazoline ring (imG14 by Tyw1), methylthiolations (ms²i⁶A37 by MiaB), and thiolations of tRNA nucleosides (s²U34 by MnmA and NcsA, s⁴U8 by Ttul, s²C32 by TtcA, s²U54 by TtuA) (Bouvier et al. 2014; Arragain et al. 2017; Chen et al. 2017; Zhou et al. 2021; He et al. 2022; Bimai et al. 2023). While several of these enzymatic systems are radical SAM-dependent, with clusters involved in the formation of a 5'Ado· radical, it has been shown that the [4Fe-4S] clusters of tRNA thiolases that catalyze the simple substitution of an oxygen atom by a sulfur atom exhibit non-redox activity and are not destroyed to serve as a sulfur donor for the thiolation reaction (Gervason et al.
2024). All known tRNA thiolation enzymes that catalyze non-redox reactions use ATP to specifically activate the target nucleotide by forming an adenylated intermediate (Fellner et al. 2018).

In non-redox tRNA thiolation reactions, the [4Fe-4S] cluster, coordinated by three residues only, likely acts as a Lewis acid to bind a sulfur atom independently of its redox state (Gervason et al. 2024). However, the possible influence of the cluster on the enzyme affinity for its tRNA substrate has not been explored yet. This is an interesting question since, if the [4Fe-4S] cluster contributes to tRNA recognition or to the flipping of the target base, this would influence the overall enzyme activity. The lack of information of this potential function of the cluster is primarily due to the extreme sensitivity of these clusters to oxygen, making it challenging to anaerobically employ classical techniques, such as electrophoretic mobility shift assays, to determine the affinity of the modification enzymes for their tRNA substrates. In this study, we address this gap by successfully determining, under anaerobic conditions, the dissociation constants ($K_d$) for tRNA of two [4Fe-4S]-dependent tRNA thiolases, which target different positions within tRNA (within the tRNA core or in the anticodon loop) by taking advantage of the fluorescence of suitably located tryptophan residues in these enzymes.

# Results and Discussion

## U8-tRNA thiolase MmTtul

The biosynthesis of s⁴U8-tRNA was attributed to enzymes initially labeled as Thil due to their association with thiamin biosynthesis in *E. coli* (Mueller et al. 1998). However, it has been established that the rhodanese-like domain (RHD) of Thil, crucial for thiamine synthesis, is not conserved in the U8-tRNA thiolase family (Martinez-Gomez et al. 2011). Consequently, it is now acknowledged that U8-tRNA sulfurases should not be designated as Thil (Bender 2011), and we have renamed them as Ttul for tRNA thiolation uridine I (He et al. 2022). Intriguingly, the synthesis of s⁴U8-tRNA in various archaea involves a [4Fe-4S]-dependent mechanism (He et al. 2022), unlike *E. coli* Ttul (EcTtul), where a persulfide-transfer mechanism is initiated by the formation of a persulfide on a crucial cysteine within the RHD (Mueller et al. 2001). Moreover, the catalytic mechanism of Ttul from species lacking the RHD, such as *Bacillus anthracis* and *Thermotoga maritima*, remains enigmatic due to the absence of conserved cysteines that
could coordinate a [Fe-S] cluster. Biochemical and spectroscopic studies have shown that U8-tRNA
thiolase from *Methanococcus maripaludis* (MmTtul) can assemble a [4Fe-4S] cluster to yield a holo-
protein capable of thiolating a tRNA transcript *in vitro* using inorganic sulfide as a sulfur source (He et al.
2022). The catalytic activity of MmTtul required the presence of the cluster along with ATP and Mg²⁺ (He
et al. 2022). Based on these findings, a mechanism akin to that of other [4Fe-4S]-dependent tRNA
thiolation enzymes (Gervason et al. 2024) has been proposed, wherein the [4Fe-4S] cluster can bind the
sulfur atom of the sulfur donor as a thiol, which serves as a the sulfur donor (He et al. 2022).

MmTtul is as an ideal system to investigate the role of a [4Fe-4S] cluster in tRNA binding through
fluorescence analysis. Indeed, this enzyme features a sole tryptophan residue (W282), which is exposed
to the surface, as evidenced by our structural model of the holo-enzyme derived from the three-
dimensional dimer of the apo-protein predicted by AlphaFold (Jumper et al. 2021) (Figure 1A).
Furthermore, W282 resides in close proximity to several positive patches on the surface of MmTtul, likely
delineating the interaction zone with tRNA (Figure 1B). Fluorescence measurements of apo-MmTtul,
following excitation at 295 nm, corresponding to tryptophan residues, revealed a spectrum with a peak
at 357 nm (Figure 1C), consistent with a highly polar environment for W282 (Vivian and Callis 2001).
Notably, denaturation of apo-MmTtul with 8 M urea did not induce any shift in the fluorescence
maximum (Figure 1C), indicating that W282 is fully solvent-exposed. We subsequently explored whether
the fluorescence of this tryptophan was responsive to the addition of tRNA substrate. As depicted in
Figure 2A, adding 4 molar equivalents of *M. maripaludis* tRNA<sup>Lys</sup> transcript (MmtRNA<sup>Lys</sup>, Figure S1A, left),
previously identified as a substrate for holo-MmTtul (He et al. 2022), to apo-MmTtul resulted in a 3-fold
decrease in fluorescence intensity compared to the apo-protein alone, accompanied by a slight shift of
the peak towards 355 nm (Figure S2A). Thus, the presence of tRNA likely alters the environment of
W282, making its fluorescence a potential probe for monitoring substrate binding to the enzyme. The
titration of apo-MmTtul with MmtRNA<sup>Lys</sup> yielded a hyperbolic decrease in fluorescence intensity (Figure
S2B), allowing us to determine a K<sub>d</sub> of 0.63 ± 0.1 µM for apo-MmTtul and MmtRNA<sup>Lys</sup> (Figure 2B, Table
1). This K<sub>d</sub> value is comparable to the reported range of 0.25 - 1.9 µM for binding of tRNA<sup>Phe</sup> to EcTtul
(Lauhon et al. 2004; Tanaka et al. 2009).
We also investigated whether the binding of the non-hydrolysable ATP analogue (α-β-methylene-adenosine-5'-triphosphate (AMP-CPP)) had any effect on the stability of the apo-MmTtul/MmtRNA<sup>Lys</sup> complex. As shown for apo-MmTtul alone, the fluorescence of apo-MmTtul was partially quenched upon MmtRNA<sup>Lys</sup> binding in the presence of AMP-CPP (Figures 2A, S2C and S2D). However, AMP-CPP did not alter the K<sub>d</sub> of apo-MmTtul for MmtRNA<sup>Lys</sup> (Table 1), as shown by the titration curve in Figure 2C.

To further substantiate our methodology, we examined the capacity of apo-MmTtul to bind a mini-RNA, which was designed based on the mini-RNAs that had been shown to be substrates of EcTtul (Lauhon et al. 2004; Tanaka et al. 2009). Indeed, the 39-nucleotides mini-RNA of *Escherichia coli* tRNA<sup>Phe</sup> corresponding to the acceptor-stem and the T-arm, called TPHE39A, was shown to bind to EcTtul with only a 2 to 4-fold increase in K<sub>d</sub> (depending on the methodology used) compared to the full-length tRNA<sup>Phe</sup> (Lauhon et al. 2004; Tanaka et al. 2009), whereas it was a substrate for EcTtul with an activity of one-third that of full-length tRNA<sup>Phe</sup> (Lauhon et al. 2004). In addition, Ttul from *Thermotoga maritima* (TmTtul) was identified as another enzyme capable of effectively catalyzing the formation of s<sup>4</sup>U8 on TPHE39A but the affinity of the enzyme/TPHE39A complex was not reported (Neumann et al. 2014). Intriguingly, the crystal structure of TPHE39A revealed significant changes in the secondary structure of the T-stem compared to that of the full-length tRNA<sup>Phe</sup> (Tanaka et al. 2009), suggesting that such conformational change in the T-stem does not disturb RNA binding. Similarly, we tested whether MmTtul could bind the 41-nucleotides mini-RNA called TLYS41A, which includes the target uridine U8, the acceptor-stem, a bulged loop and the T-stem loop of MmtRNA<sup>Lys</sup> (Figure S1A, right). Indeed, fluorescence analysis indicated that apo-MmTtul can interact with TLYS41A, albeit with a three-fold lower affinity compared to full-length MmtRNA<sup>Lys</sup> (Table 1, Figure S3).

Previous studies have highlighted the importance of magnesium ions, in addition to the [4Fe-4S] cluster and ATP, for MmTtul to be catalytically active (He et al. 2022). According to our model of the complex of MmTtul with ATP and Mg<sup>2+</sup> (Figures 1A and 1B), the Mg<sup>2+</sup> binding site is predicted to be located near the tRNA binding site. Consequently, we investigated the impact of Mg<sup>2+</sup> ions on the binding of both TLYS41A and the full-length MmtRNA<sup>Lys</sup> to apo-MmTtul by fluorescence (Figure S3). Our results
showed a 3.8-fold and 3.5-fold decrease in $K_d$, for TLYS41A and MmtRNA<sup>Lys</sup>, respectively, in the presence of $Mg^{2+}$ (Table 1).

Next, cluster reconstitution was performed with apo-MmTtul to generate holo-MmTtul and investigate the potential role of the cluster in tRNA binding. The UV/visible absorption spectrum of the protein after [Fe-S] cluster reconstitution revealed a characteristic peak around 410 nm consistent with the presence of a [4Fe-4S] cluster bound within holo-MmTtul (Figure S4A). The iron quantification (3.2 Fe atoms per monomer) together with the 410/280 nm absorbance ratio of 0.26 indicated that the protein underwent successful cluster reconstitution in the [4Fe-4S] cluster state, as reported previously (He et al. 2022). As shown in Figure S5A, holo-MmTtul catalyzed the formation of $s^4U$ in the tRNA<sup>Lys</sup> transcript. Interestingly, we found that holo-MmTtul also modifies TLYS41A (Figure S5A), but with an activity approximately six times lower than that observed with the full-length tRNA. Nevertheless, this indicates that a mini-RNA is a substrate for MmTtul, as previously reported for EcTtul and TmTtul (Lauhon et al. 2004; Tanaka et al. 2009).

We then applied our fluorescence approach to investigate the binding of tRNA to holo-MmTtul. The fluorescence spectrum of the holo-protein displayed a peak maximum at 344 nm, indicating a significant 9 nm blue shift compared to the apo-protein (Figure 2D). This finding suggests that the presence of the [Fe-S] cluster creates a less polar environment for W282. As with apo-MmTtul, the addition of MmtRNA<sup>Lys</sup> reduced the fluorescence of holo-MmTtul, regardless of the presence of AMP-CPP (Figure 2D). Moreover, the analysis of the isotherms for tRNA binding to holo-MmTtul, depicted by the normalized fluorescence change at 355 nm as a function of tRNA<sup>Lys</sup> concentrations, indicated that, similar to apo-MmTtul, AMP-CPP did not significantly alter the $K_d$ for tRNA<sup>Lys</sup>, which remained around 0.5-0.7 µM (Figures 2E & 2F, Table 1). Altogether, apo- and holo-MmTtul have a comparable affinity for MmtRNA<sup>Lys</sup> (Table 1), in the presence or absence of AMP-CPP, suggesting that the [4Fe-4S] cluster has no discernible effect on the overall binding of tRNA.

We subsequently questioned whether the [Fe-S] cluster could have a local effect on the recognition of U8 by Ttul enzymes. Indeed, analysis of the X-ray structures of unbound tRNAs (Fernández-Millán et al. 2016) indicates that the U8 position is not readily accessible to solvent,
suggesting the necessity of a conformational change of the U8-containing region and/or the flipping out
of the target base by Ttul to facilitate its thiolation. To investigate this possibility, we replaced adenine 9
with a 2-aminopurine (2AP) in MmtRNA<sup>Lys</sup> (2AP9-MmtRNA<sup>Lys</sup>)(Figure 3A) and first examined whether this
modified tRNA could serve as a substrate for MmTtul. Interestingly, as shown in Figure S5A, MmTtul not
only modifies 2AP9-MmtRNA<sup>Lys</sup> but the activity is enhanced when 2AP is present at position 9 instead of
adenine. 2AP is expected to undergo fluorescence quenching upon base stacking, and its unstacking to
induce a significant increase in fluorescence. Indeed, substituting adenine with 2AP was shown to
facilitate the study of the flipping mechanism of the A57,A58-tRNA methyltransferase Trml from
*Pyrococcus abyssi*, with the flipping of adenine 57 by Trml resulting in a 2.5-fold enhancement in 2AP
fluorescence (Hamdane et al. 2014). Thus, we monitored the effect of the addition of MmTtul to 2AP-
tRNA<sup>Lys</sup> on the fluorescence of this fluorophore when excited at 320 nm. As depicted in Figure 3B, the
addition of a 4-fold molar excess of apo-MmTtul to 2AP9-MmtRNA<sup>Lys</sup> resulted in a 30% increase in 2AP
fluorescence. Adding the same quantity of holo-MmTtul to 2AP9-MmtRNA<sup>Lys</sup> produced a comparable
25% enhancement in 2AP fluorescence. The observation of a 9 nm red shift in the maximum fluorescence
in the presence of the [Fe-S] cluster suggests a more polar environment for 2AP9 when bound to holo-
MmTtul rather than to apo-MmTtul. Due to its positive charge, the cluster likely generates a local electric
field that alters the electronic environment of 2AP. In an effort to rationalize the relatively modest
increase in fluorescence of 2AP9-MmtRNA<sup>Lys</sup> upon binding MmTtul, we generated an AlphaFold3 model
of MmtRNA<sup>Lys</sup> and analyzed the environment of U8. As shown in Figure 3C, the U8 and A9 bases are
separated by the U13 base so that the change of fluorescence of 2AP9 upon binding of MmTtul cannot
be directly related to the flipping of U8 but rather to a conformational change of the U8-U13 loop.
Interestingly, given that the binding of 2AP9-MmtRNA<sup>Lys</sup> to apo-MmTtul and holo-MmTtul is similar
(Figure 3B), the presence of the [4Fe-4S] cluster does not significantly alter the conformational changes
around A9 occuring upon binding MmTtul. Since 2AP9-MmtRNA<sup>Lys</sup> is a good substrate of MmTtul (Figure
S5A), flipping of U8 likely occurs but the experimental structure of the holo-MmTtul/2AP9-MmtRNA<sup>Lys</sup>
complex is needed to validate this hypothesis.
C32-tRNA thiolase EcTtcA

We used TtcA from *Escherichia coli* (EcTtcA), a [4Fe-4S]-dependent C32-tRNA thiolase (Bouvier *et al. 2014*) to expand our investigation on the role of the iron-sulfur cluster in tRNA thiolation enzymes. TtcA enzymes catalyze the insertion of sulfur within C32, next to the anticodon loop, in tRNAs (Figure S1B), in the presence of ATP. Unlike MmTtul, EcTtcA possesses two tryptophan residues, W231 and W291 (Figure 1D) that are located near the putative tRNA binding site, according to their location next to a positive region of the electrostatic interface (Figure 1E). The s²C32 modification is present in four *E. coli* tRNAs (Cappannini *et al. 2024*), including tRNAArg (ICG) (EctRNAArg) (Figure S1B, left). It was previously shown that the [4Fe-4S] cluster and ATP were essential for s²C biosynthesis by EcTtcA within a bulk tRNA isolated from an *E. coli* ΔttcA strain, using cysteine desulfurase IscS and cysteine, in the presence of dithiothreitol, as the sulfur source (Bouvier *et al. 2014*). First, we could efficiently reconstitute a [4Fe-4S] cluster in EcTtcA, as shown by the UV-visible spectrum of holo-EcTtcA (Figure S4B), in agreement with the quantification of Fe (3.6 atoms per monomer). The holo-enzyme was active on an EctRNAArg transcript but also on a 17-nucleotides anticodon loop called mini-RNAArg (Figure S1B, right), in the presence of ATP and inorganic sulfide as the sulfur source (Figure S5B).

We determined the K_d of the apo- and holo-forms of EcTtcA for the EctRNAArg transcript by monitoring protein fluorescence, as detailed previously for MmTtul. Fluorescence analysis revealed that the addition of EctRNAArg transcript, to either apo- or holo-EcTtcA, induced fluorescence quenching in a concentration-dependent manner (Figure 4). The resulting titration isotherms yielded similar K_d values for the apo- and holo-forms (3.5 and 4 µM, respectively, Table 1), suggesting that, as with MmTtul, the [4Fe-4S] cluster did not significantly affect tRNA binding by EcTtcA.

In conclusion, we report here the first measurement of the affinity of two [4Fe-4S]-dependent tRNA modifying enzymes for their tRNA substrates, in the apo, and holo states under anaerobic conditions. Our results show that the [4Fe-4S] clusters of MmTtul and EcTtcA play no obvious role in tRNA binding. Additionally, it is likely that the cluster of MmTtul is not involved in flipping the target U8 base for modification, a process that appears to be driven by the enzyme itself. These findings suggest
an exclusively chemical role of the [4Fe-4S] cluster in the thiolation of tRNA. Finally, our results regarding two tRNA thiolation enzymes possessing tryptophan residues located near the tRNA binding site have unveiled a robust fluorescence method for determining K<sub>d</sub> values of these enzymes for their RNA substrates under various conditions, which could be extended both to other [Fe-S]-dependent or [Fe-S]-independent RNA modification enzymes to determine their affinity constants for their RNA substrate.

## Material and methods

The non-hydrolyzable ATP analogue AMP-CPP (α-β-methylene-adenosine-5'triphosphate) was bought from Jenabioscience and the RNA transcripts from Horizon Discovery Biosciences.

### Heterologous gene overexpression and aerobic purification of MmTtul and EcTtcA

Apo-MmTtul was produced and purified as previously described (He et al. 2022). The gene coding for EcTtcA was synthesized by Genscript and cloned between the Ndel and BamHI sites in the pET15b plasmid, in order to add a cleavable N-terminal histidine tag to the protein. Apo-EcTtcA was produced and purified after cleavage of the histidine tag by the H3C protease according to (He et al. 2022).

### In vitro [Fe-S] cluster reconstitution and anaerobic purification of holo-MmTtul and holo-EcTtcA

The reconstitution of the [4Fe-4S] cluster and purification of holo-MmTtul and holo-EcTtcA were performed in a glove box (MBraun) containing less than 0.5 ppm O<sub>2</sub>. After incubation of as-purified proteins (100 µM) with 15 mM dithiothreitol for 15 min, a 5-fold molar excess of ferrous ammonium sulfate and sodium sulfide was added. Cluster reconstitution was followed by the appearance of a band at around 410 nm in the UV-visible spectrum, which was recorded with a XL-100 UVICON spectrophotometer equipped with optical fibers, and incubation was stopped after 3 h. After centrifugation for 10 min at 20,000 g, holo-MmTtul and holo-EcTtcA were loaded onto a Superdex 200 Increase 10/300 GL gel filtration column (Cytiva) equilibrated in 25 mM HEPES pH 7.5, 150 mM NaCl. The Fe content in the holo-proteins after cluster reconstitution was quantified using the Fish method (Fish 1988).
Activity tests

The RNA transcripts were refolded by heating for 2 min at 85°C and then cooling on ice for 4 min. Holo-MmTtul and holo-EcTtcA (5 µM) were incubated with their full-length or mini-RNA transcripts (15 µM) for one hour at 37°C in 50 µl 20 mM HEPES pH 7.5, 150 mM NaCl in the presence of 0.25 mM ATP, 2.5 mM MgCl₂ and 1 mM Na₂S in an MBraun glove box. The reaction was stopped by adding 1 µl of 3 M formic acid, and the pH adjusted to around 6.5 by adding 3 µl of 1 M Tris pH 8.5. After reaction, tRNA was digested and s⁴U or s²C was separated by HPLC-MS/MS and quantified using synthetic s⁴U or s²C standard as described previously (Bimai et al. 2023).

Fluorescence studies

The emission fluorescence spectra were recorded in a quartz cuvette at 37°C on a Cary eclipse fluorescence spectrophotometer (Varian) in an MBraun glove box. After exciting at the Trp wavelength of 295 nm or at 320 nm for experiments with 2AP-containing RNA, the fluorescence emission was recorded every nm from 335 to 550 nm. The excitation and emission slits were set to 5 and 10 nm, respectively. In all experiments, enzyme (1 µM) was incubated with RNA transcript (from 0 to 3.2 µM) in 700 µl of 25 mM HEPES pH 7.5, 150 mM NaCl, 3 mM MgCl₂ in the presence or absence of AMP-CPP (3 mM). MgCl₂ was omitted only when the effect of MgCl₂ on tRNA binding was studied (Figure S3). Before recording the spectra, enzyme and refolded RNA were incubated for 5 min to reach the equilibrium. The resulting spectra were corrected for the contribution of the buffer. ΔF<sub>N</sub> (Normalized value of fluorescence) = (ΔF at a given RNA concentration)/(ΔF_max).

Structural modeling

The AlphaFold models of MmTtul and EcTtcA were calculated with the Google Colab platform and
AlphaFold2_advanced option

<https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/beta/AlphaFold2_advanced.ipynb#scrollTo=ITcPnLkLuDDE> that does not use templates (homologous structures), and refined using the Amber-relax option to enhance the accuracy of the side chains geometry. The default mode of
sampling options was used: num_models=5, ptm option, num_ensemble=1, maxala=3, tol=0,

numSamples=1, 1:1 value as the homo-oligomer assembly option. The models were ranked according
to their pLDDT confidence values (between 0 and 100, from low to high confidence).

The models of holo-MmTtul and holo-EcTtcA were built by taking the position of the cluster of holo-
MmNcsA (PDB code 6SCY) (Bimai et al. 2023) after superposition of its ATPase region with that of the
MmTtul and EcTtcA AlphaFold models, respectively. Similarly, the ATP and Mg<sup>2+</sup> positions of TmTtul in
complex with ATP and Mg<sup>2+</sup> (PDB code 4KR7), after superposition of TmTtul with the AlphaFold model of
MmTtul, were used to build the complex of MmTtul with ATP and Mg<sup>2+</sup>.

Acknowledgments

This work was supported by the Centre National de la Recherche Scientifique, Sorbonne Université as
well as the “Initiative d’Excellence” program from the French State (Grant “DYNAMO”, ANR-11-LABX-
0011-01) and the French National Research Agency (Grant sulfo-tRNA, ANR-22-CE44-0012). We thank
Nisha He for preparing apo-MmTtul.

Competing financial interests

The authors declare no competing financial interests.
<table><caption>**Table 1. K<sub>d</sub> values of the protein/RNA complexes studied**</caption><thead><tr><td>complex</td><td>K<sub>d</sub> (μM)*</td><td>Figure</td></tr></thead><tbody><tr><td>apo-MmTtul/MmtRNA<sup>Lys</sup></td><td>0.63 ± 0.1</td><td>Fig 2B</td></tr><tr><td>apo-MmTtul/MmtRNA<sup>Lys</sup> +AMP-CPP</td><td>0.55 ± 0.08</td><td>Fig 2C</td></tr><tr><td>apo-MmTtul/MmtRNA<sup>Lys</sup> no Mg<sup>2+</sup></td><td>2.2 ± 0.4</td><td>Fig S3B</td></tr><tr><td>apo-MmTtul/TLYS41A</td><td>1.9 ± 0.1</td><td>Fig S3A</td></tr><tr><td>apo-MmTtul/TLYS41A no Mg<sup>2+</sup></td><td>7.2 ± 1.4</td><td>Fig S3A</td></tr><tr><td>holo-MmTtul/MmtRNA<sup>Lys</sup></td><td>0.5 ± 0.13</td><td>Fig 2E</td></tr><tr><td>holo-MmTtul/MmtRNA<sup>Lys</sup>+AMP-CPP</td><td>0.7 ± 0.25</td><td>Fig 2F</td></tr><tr><td>apo-EcTtcA/EctRNA<sup>Arg</sup></td><td>3.5 ± 0.6</td><td>Fig 4</td></tr><tr><td>holo-EcTtcA/EctRNA<sup>Arg</sup></td><td>4 ± 1.5</td><td>Fig 4</td></tr></tbody></table>

<footnote>*The standard deviation values were calculated from 3 and 2 replicates for the experiments performed with the apo- and holo-proteins, respectively.</footnote>
Figures

**Figure 1.** Environment of tryptophan residues in MmTtul and EcTtcA. (A) Alphafold2 model of the holo-MmTtul dimer in complex with ATP and Mg²⁺. One monomer is colored in cyan and the other in blue. Residues W282 and ATP are depicted as sticks (in magenta and red, respectively), while the [4Fe-4S] cluster and Mg²⁺ ions are shown in sphere representation with sulfur atoms colored in yellow, iron atoms in orange, and Mg atoms in black. (B) Electrostatic surface of the holo-MmTtul dimer (negative charge in red, positive charge in blue). (C) Fluorescence spectra obtained at λ<sub>ex</sub> = 295 nm of native apo-MmTtul (in green) and after denaturation with 8 M urea (in red).

(D) Alphafold2 model of the holo-EcTtcA dimer. One monomer is colored in cyan and the other in blue. Residues W231 and W291 are depicted as red sticks, while the [4Fe-4S] cluster is shown in sphere representation. (E) Electrostatic surface of the holo-EcTtcA dimer.

<figure><img src="image_2.png" /></figure>
<caption>**Figure 2.** Monitoring of MmtRNA<sup>Lys</sup> binding to apo- and holo-MmTtul by fluorescence. (A) Fluorescence spectra obtained at λ<sub>ex</sub> = 295 nm of apo-MmTtul (0.5 µM) alone (in green), apo-MmTtul + 3 mM AMP-CPP (in red), apo-MmTtul + 2 µM tRNA<sup>Lys</sup> (in blue), apo-MmTtul + 3 mM AMP-CPP + 2 µM tRNA<sup>Lys</sup> (in magenta). (B) and (C) Saturation curves of W282 fluorescence at 355 nm normalized to tRNA<sup>Lys</sup> concentration for apo-MmTtul in the absence (B) and presence of 3 mM AMP-CPP (C). (D) Fluorescence spectra obtained at λ<sub>ex</sub> = 295 nm of holo-MmTtul (0.5 µM) alone (in green), holo-MmTtul + 3 mM AMP-CPP (in red), holo-MmTtul + 2 µM tRNA<sup>Lys</sup> (in blue), holo-MmTtul + 3 mM AMP-CPP + 2 µM tRNA<sup>Lys</sup> (in magenta). (E) and (F) Normalized saturation curves of fluorescence at 355 nm, for holo-MmTtul in the absence (E) and presence of 3 mM AMP-CPP (F).</caption>

A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /></figure>

C

<figure><img src="image_5.png" /></figure>

D

<figure><img src="image_6.png" /></figure>

E

<figure><img src="image_7.png" /></figure>

F

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>**Figure 3.** Sensitivity of the [4Fe-4S] cluster of MmTtul to the environment of the U8 target base in MmtRNA<sup>Lys</sup>. (A) Chemical structure of 2-aminopurine (2AP) and its position (in black) in 2AP9-MmtRNA<sup>Lys</sup>, adjacent to the target base U8 (in red). (B) Fluorescence spectra obtained at λ<sub>ex</sub> = 320 nm of 2AP9-MmtRNA<sup>Lys</sup> (0.5 µM). 2AP9-MmtRNA<sup>Lys</sup> alone (in green), in the presence of apo-MmTtul (2 µM) + 3 mM AMP-CPP (in blue) or in the presence of holo-MmTtul (2 µM) + 3 mM AMP-CPP (in red). Apo-MmTtul in the absence of 2AP9-MmtRNA<sup>Lys</sup> (in black). (C) Region surrounding U8 in the AlphaFold3 model of MmtRNA<sup>Lys</sup> (pTM score of 0.44). The target base U8 is shown in magenta.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Figure 4.** Monitoring of EctRNA<sup>Arg</sup> binding to apo- and holo-EcTtcA by fluorescence. (A) and (B) Normalized saturation curves of fluorescence at 343 nm for apo-EcTtcA (A) and holo-EcTtcA (B).</figcaption></figure>

<figure><img src="image_11.png" /></figure>
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