ArcS from Thermococcus kodakarensis transfers L-lysine to preQ0 nucleoside derivatives as minimum substrate RNAs

Shu Fujita; Yuzuru Sugio; Takuya Kawamura; Ryota Yamagami; Natsuhisa Oka; Akira Hirata; Takashi Yokogawa; Hiroyuki Hori

doi:10.1016/j.jbc.2024.107505

. 2024 Jun 27;300(8):107505. doi: 10.1016/j.jbc.2024.107505

ArcS from Thermococcus kodakarensis transfers L-lysine to preQ₀ nucleoside derivatives as minimum substrate RNAs

Shu Fujita ¹, Yuzuru Sugio ¹, Takuya Kawamura ¹, Ryota Yamagami ¹, Natsuhisa Oka ^2,^3,⁴, Akira Hirata ⁵, Takashi Yokogawa ^2,^4,⁶, Hiroyuki Hori ^1,^∗

PMCID: PMC11298593 PMID: 38944122

Abstract

Archaeosine (G⁺) is an archaea-specific tRNA modification synthesized via multiple steps. In the first step, archaeosine tRNA guanine transglucosylase (ArcTGT) exchanges the G15 base in tRNA with 7-cyano-7-deazaguanine (preQ₀). In Euryarchaea, preQ₀15 in tRNA is further modified by archaeosine synthase (ArcS). Thermococcus kodakarensis ArcS catalyzes a lysine-transfer reaction to produce preQ₀-lysine (preQ₀-Lys) as an intermediate. The resulting preQ₀-Lys15 in tRNA is converted to G⁺15 by a radical S-adenosyl-L-methionine enzyme for archaeosine formation (RaSEA), which forms a complex with ArcS. Here, we focus on the substrate tRNA recognition mechanism of ArcS. Kinetic parameters of ArcS for lysine and tRNA-preQ₀ were determined using a purified enzyme. RNA fragments containing preQ₀ were prepared from Saccharomyces cerevisiae tRNA^Phe-preQ₀15. ArcS transferred ¹⁴C-labeled lysine to RNA fragments. Furthermore, ArcS transferred lysine to preQ₀ nucleoside and preQ₀ nucleoside 5′-monophosphate. Thus, the L-shaped structure and the sequence of tRNA are not essential for the lysine-transfer reaction by ArcS. However, the presence of D-arm structure accelerates the lysine-transfer reaction. Because ArcTGT from thermophilic archaea recognizes the common D-arm structure, we expected the combination of T. kodakarensis ArcTGT and ArcS and RaSEA complex would result in the formation of preQ₀-Lys15 in all tRNAs. This hypothesis was confirmed using 46 T. kodakarensis tRNA transcripts and three Haloferax volcanii tRNA transcripts. In addition, ArcTGT did not exchange the preQ₀-Lys15 in tRNA with guanine or preQ₀ base, showing that formation of tRNA-preQ₀-Lys by ArcS plays a role in preventing the reverse reaction in G⁺ biosynthesis.

Keywords: archaeosine, RNA modification, ArcTGT, ArcS, RaSEA

To date, more than 100 modified nucleosides have been identified in tRNA (1, 2, 3). Almost all modified nucleosides in tRNA are synthesized from A, G, C, or U by chemical decorations (1, 2, 3). However, 7-deazaguanine derivatives are distinct because these modified nucleosides are initially introduced into tRNA by a base exchange reaction. Archaeosine (G⁺) contains 7-deazaguanine: G⁺ is 7-formamidino-7-deazaguanosine (2-amino-4, 7-dihydro-4-oxo-7-β-D-ribofuranosyl-1H-pyrro [2, 3-days] pyrimidine-5- carboximidamide) (4) (Fig. 1).

**The G**⁺**biosynthesis pathway of *T. kodakarensis*.** ArcTGT catalyzes the base exchange reaction from G15 to preQ₀15 in tRNA. The resultant preQ₀15 in tRNA is further modified to preQ₀-Lys by ArcS. Finally, the synthesized preQ₀-Lys in tRNA is converted to G⁺ by RaSEA.

G⁺ was first identified at position 15 in elongator tRNA^Met from Thermoplasma acidophilum, a thermo-acidophilic archaeon (5, 6), as an unknown modified nucleoside (7). It was then found in tRNAs from numerous archaea including Haloferax volcanii (8), Thermoproteus neutrophilus (9), Sulfolobus acidocaldarius (4, 10, 11), Methanococcus maripuldis (10, 11), Pyrococcus furiosus (10, 11), Haloarcula marismortui (12, 13), Methanocaldococcus jannaschii (14), Sulfurisphaera tokodaii (15), and Thermococcus kodakarensis (16, 17, 18, 19, 20). Furthermore, tRNA^Leu from T. acidophilum exceptionally possesses 2 G⁺ modifications at positions 13 and 15 (16, 21), while initiator tRNA^Met of this archaeon possesses only G⁺15 (22, 23).

A bioinformatics study predicted that G⁺15 stabilizes the L-shaped tRNA structure through reinforcement of the G15-C48 tertiary base pair (24). Consistent with this, the presence of G⁺ in the T. kodakarensis tRNA^Gln transcript increases the melting temperature in the presence of 100 μM and 10 mM MgCl₂ (20). Furthermore, in T. kodakarensis, a deletion strain of the arcTGT gene, which encodes archaeosine tRNA guanine transglucosylase (ArcTGT), an enzyme for the first step of G⁺ synthesis in tRNA cannot grow at high temperatures (17). These studies reveal that the G⁺15 modification in tRNA stabilizes the L-shaped tRNA structure and is required for the survival of thermophilic archaea at high temperatures.

The biosynthetic pathway of G⁺ in tRNA consists of multiple steps (Fig. 1). In the first step, ArcTGT exchanges the G15 base in tRNA with 7-cyano-7-deazaguanine (preQ₀) (25, 26). ArcTGT proteins and their genes have been experimentally identified in several archaea including H. volcanii (25, 26), M. janaschii (27), Pyrococcus horikoshii (28, 29, 30), P. furiosus (31), Methanosarcina barkeri (31), Methanosarcina acetivorans (32, 33), T. acidophilum (16), and T. kodakarensis (16), which is consistent with the wide spread of G⁺ in archaeal tRNAs. The enzymatic properties and tRNA recognition mechanisms of ArcTGT from several archaea have been reported (16, 25, 26, 27, 28, 31, 32, 33). Furthermore, crystal structure studies of ArcTGT and the complex between ArcTGT and tRNA have been performed (29, 30).

The second and subsequent steps of the G⁺ biosynthesis pathway differ between archaea species. In Euryarchaea, preQ₀15 in tRNA is further modified by archaeosine synthase (ArcS) (Fig. 1). ArcS was initially found by amino acid sequence homology with ArcTGT (34) and then analysis of tRNAs from a H. volcanii arcS gene deletion strain revealed that ArcS is involved in G⁺ biosynthesis (34). ArcS from M. janaschii uses NH₄⁺, asparagine, or glutamine as a nitrogen source and directly synthesizes G⁺15 from preQ₀ in tRNA (34). In contrast, ArcS proteins from T. acidophilum, M. acetivorans,and T. kodakarensis catalyze a lysine-transfer reaction and synthesize preQ₀-lysine (preQ₀-Lys) at position 15 in tRNA as an intermediate in G⁺ biosynthesis (19) (Fig. 1). ArcS proteins from T. acidophilum, M. acetivorans,and T. kodakarensis do not use NH₄⁺, asparagine, or glutamine as a nitrogen source (19). The resulting preQ₀-Lys15 in tRNA is converted to G⁺15 by a radical S-adenosyl-L-methionine enzyme for archaeosine formation (RaSEA) (19). Because 196 Euryarchaea species including M. janaschii possess the gene set of arcS and RaSEA, the latter pathway (combination of ArcS and RaSEA) may be mainly used in the G⁺15 formation in living cells (19). In contrast, most Crenarchaea do not possess the arcS gene (35). In the case of Pyrobaculum calidifontis (a Crenarchaea), a QueF-like protein synthesizes G⁺15 in tRNA from preQ₀15 using NH₄⁺ as a nitrogen donor (36, 37).

These progresses described above in the study of G⁺ formation in tRNA have been achieved over the last 3 decades. However, the mechanism of substrate tRNA recognition by ArcS is still unknown. To address this issue, we have performed biochemical analyses which are described in this study.

Results

Purification of recombinant ArcTGT, ArcS, and RaSEA complex

To measure the enzymatic activity of ArcS, tRNA containing preQ₀ at position 15 (tRNA-preQ₀15), which is prepared using ArcTGT, is required as a substrate. However, ArcTGT from T. acidophilum is not expressed as a soluble protein in Escherichia coli cells (16). Furthermore, because ArcTGT from M. acetivorans is a split-type (32), the expression of two protein subunits in E. coli cells is required. The lysine-transfer activity of ArcS from T. kodakarensis was confirmed in our previous report (19). In contrast, ArcS from M. janaschii uses NH₄⁺, asparagine, or glutamine as a nitrogen source (34). Therefore, we used the set of ArcTGT, and ArcS and RaSEA complex from T. kodakarensis in this study. ArcS from T. kodakarensis forms a complex with RaSEA (19). Although recombinant ArcS protein expressed in E. coli cells is soluble in the absence of RaSEA, this free ArcS protein does not catalyze the lysine-transfer reaction (19). Only the complex of ArcS and RaSEA demonstrates the lysine-transfer activity (19). Furthermore, ArcTGT does not form a complex with ArcS. To show this, we performed a co-expression experiment in E. coli (Fig. S1). Three proteins (ArcTGT, His x 6-ArcS, and RaSEA) were co-expressed in E. coli cells, co-purified by NiNTA Superflow column chromatography, and analyzed by 10% SDS-PAGE. RaSEA was co-purified with His x 6-ArcS, showing that ArcS and RaSEA form a complex. In contrast, ArcTGT was not purified with His x 6-ArcS. Thus, this experimental result shows that ArcS does not form a complex with ArcTGT. T. kodakarensis ArcTGT and ArcS and RaSEA complex were expressed in E. coli cells and purified as shown in Fig. S2, A and B. It should be mentioned that these enzymes were purified under aerobic conditions. Thus, RaSEA is in an apo form, which does not contain a Fe-S cluster. Therefore, preQ₀-Lys15 in tRNA is not converted to G⁺15 in the reaction mixture containing ArcS and RaSEA complex, and formation of preQ₀-Lys15 in tRNA by ArcS can be directly monitored.

Measurement of kinetic parameters of lysine transfer reaction

ArcS was originally found via its amino acid sequence homology with ArcTGT (34). Initially, therefore, we expected that the substrate tRNA recognition mechanism of ArcS might resemble that of ArcTGT. Fortunately, the substrate tRNA recognition mechanism of ArcTGT from a thermophilic archaeon (P. horikoshii) has been reported (28). ArcTGT from P. horikoshii does not recognize the L-shaped tRNA structure and nucleotide sequences (except for G15) in the D-loop but rather recognizes the ribose-phosphate backbone of the D-arm (28). However, the three-dimensional structure of tRNA from T. kodakarensis has not been reported. Therefore, we selected Saccharomyces cerevisiae tRNA^Phe-preQ₀15 (Fig. 2A) as a model substrate because the three-dimensional structure of this tRNA is well established (38). It should be mentioned that native tRNA^Phe from S. cerevisiae does not possess the G⁺15 modification (38). The substrate tRNA^Phe transcript, in which preQ₀ was present at position 15, was prepared using the base exchange reaction of ArcTGT. We measured the kinetic parameters of ArcS for lysine (Fig. 2B) and tRNA^Phe-preQ₀15 (Fig. 2C). The values of kinetic parameters are given in Table 1. The Km value of ArcS for lysine was determined to be 40.9 μM (Fig. 2B), which is comparable to that of E. coli and S. cerevisiae lysyl-tRNA synthetases for lysine (27 μM and 38 μM, respectively; ref. (39, 40)). Although the concentration of lysine in T. kodakarensis cells is unknown, this Km value of ArcS is normal for a lysine-related enzyme. In contrast, the Km value of ArcS for tRNA^Phe-preQ₀15 was determined to be 12.3± 3.2 μM (Fig. 2C and Table 1). This value is considerably large for the Km value for substrate tRNA of a tRNA modification enzyme. For example, the Km value of P. horikoshii ArcTGT for tRNA^Val transcript has been reported to be 0.57 μM (28). We suspected that the large Km value for tRNA^Phe-preQ₀ might be caused by the sequence of S. cerevisiae tRNA^Phe. As described above, S. cerevisiae tRNA^Phe-preQ₀ is an artificial substrate for ArcS. To address this issue, we prepared T. kodakarensis tRNA^Trp-preQ₀ transcript and performed the kinetic studies using this tRNA transcript (Fig. 2D). In T. kodakarensis tRNAs, the presence of G⁺15 has only been confirmed in tRNA^Trp (18). As shown in Figure 2E and Table 1, the Km value for T. kodakarensis tRNA^Trp-preQ₀ transcript was determined to be 54.9 μM, which is comparable to that for S. cerevisiae tRNA^Phe-preQ₀ transcript. Thus, the large Km values of ArcS are common to both artificial and natural substrate tRNAs. However, the Kcat values of ArcS for S. cerevisiae tRNA^Phe-preQ₀ and T. kodakarensis tRNA^Trp-preQ₀ were determined to be around 3.7 min⁻¹ and 7.5 min⁻¹, respectively. These Kcat values for lysine-transfer reaction by ArcS are larger than that of tRNA-preQ₀15 formation by ArcTGT: the Kcat of P. horikoshii ArcTGT is calculated to be 4.9 min⁻¹ from the kinetic values reported (28). Therefore, the velocity of the lysine-transfer reaction mediated by ArcS may be sufficient for tRNA-preQ₀-Lys15 formation in living cells.

Table 1.

Kinetic parameters for lysine and substrate RNAs

Substrate	Km [μmol]	Kcat [min⁻¹]	Relative Kcat/Km
L-lysine	40.9 ± 8.7	6.1 ± 0.7
S. cerevisiae tRNA^Phe-preQ₀	12.3 ± 3.2	3.7 ± 0.4	1.00
T. kodakarensis tRNA^Trp-preQ₀	54.9 ± 23.4	7.5 ± 0.9	0.45
21 nt RNA-preQ₀ fragment	18.4 ± 7.4	14.8 ± 1.8	2.67
64 nt RNA-preQ₀ fragment	28.3 ± 6.2	27.1 ± 1.9	3.18
5′ P-preQ₀ nucleotide	433.4 ± 6.0	23.2 ± 3.7	0.18

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Relative Kcat/Km value for S. cerevisiae tRNA^Phe is expressed as 1.00.

ArcS does not require the L-shaped tRNA structure for the lysine-transfer reaction

To clarify the recognition site(s) of ArcS in tRNA, we prepared RNA fragments derived from tRNA^Phe-preQ₀15 by deoxyribozyme (DNAzyme) reaction (41) (Fig. 3A). We selected 8-17 DNAzyme (42) for this study because it is able to cleave AG sequences in RNAs site-specifically (42, 43). We designed two 8-17 DNAzymes and cleaved tRNA^Phe-preQ₀15: one cleaves between A21 and G22 and the other cleaves between A64 and G65 (Fig. 3B). The resultant preQ₀-RNA fragments (21 nt and 64 nt) were purified by 10% polyacrylamide gel containing 7 M urea electrophoresis [10% PAGE (7 M urea)]. We used full-length tRNA^Phe-G15 and tRNA^Phe-preQ₀15 transcripts as negative and positive controls, respectively. These RNAs (8.5 μM each) were incubated with 0.11 μM ArcS-RaSEA complex and 100 μM ¹⁴C-labeled lysine at 60 °C for 10 min. The RNAs were treated with phenol-chloroform, recovered by ethanol precipitation, and then 0.03 A260 units of RNAs were analyzed by 10% PAGE (7 M urea) (Fig. 3C left). The autoradiogram of the same gel was collected (Fig. 3C right). Under the condition tested, the band intensities show relative velocities of ¹⁴C-lysine-transfer to RNAs. As shown in Figure 3C right, ¹⁴C-lysine was transferred to all RNAs containing preQ₀. In contrast, ¹⁴C-lysine was not transferred to full-length tRNA^Phe-G15 (negative control). These results clearly show that ArcS does not require the L-shaped tRNA structure for the lysine-transfer reaction. Furthermore, to our surprise, the band intensities in the autoradiogram showed that the incorporation of ¹⁴C-lysine into the 21 nt and 64 nt RNA fragments was more efficient than that into the full-length tRNA-preQ₀15. To confirm this, we performed kinetic studies using the 21 nt and 64 nt RNA fragments (Fig. 3, D and E, and Table 1). As shown in Figure 3D and Table 1, both the Km and Kcat values for the 21 nt RNA fragment were improved relative to those for full-length tRNA^Phe-preQ₀15. Thus, this analysis reveals that the L-shaped tRNA structure has a negative effect on the lysine-transfer reaction of ArcS. Furthermore, in the 21 nt RNA fragment, the D-arm structure as well as the L-shaped tRNA structure is disrupted. Therefore, it is also clear that the D-arm structure is not essential for the lysine-transfer reaction by ArcS. However, the Km and Kcat values for the 64 nt RNA fragment revealed that the 64 nt RNA fragment was the best substrate of the RNAs tested (Table 1). In the 64 nt RNA fragment, the D-arm structure is not disrupted. These results show that the presence of a D-arm structure accelerates the velocity of the lysine-transfer reaction by ArcS although the D-arm structure is not essential for the lysine-transfer reaction.

ArcS can transfer lysine to preQ₀ nucleoside and the presence of 5′-phosphate accelerates the reaction

ArcS can transfer lysine to a 21 nt RNA fragment containing preQ₀. This result prompted us to investigate the identity of the minimum substrate RNA for ArcS (Fig. 4). Because preQ₀ and its derivatives absorb ultra-violet light at 305 nm (UV_305nm), the formation of lysine-adducts can be monitored by absorbance of UV_305nm using high-performance liquid column (HPLC) chromatography. The elution time of preQ₀ and its derivatives were determined using standard compounds, which were prepared by organic chemistry or nuclease digestion of full-length tRNA^Phe-preQ₀-Lys (details are described in Experimental Procedures). When preQ₀ base was used as a substrate, ArcS did not transfer lysine to preQ₀ base (Fig. 4A lower). It should be mentioned that the peak at 25.8 min (marked by an asterisk in Fig, 4) is a result of the presence of 2-mercaptoethanol in the reaction mixture. Therefore, this peak was also observed in the negative control, to which ArcS was not added (Fig. 4A middle). When 34.0 μM preQ₀ nucleoside was incubated with 0.23 μM ArcS and 200 μM lysine at 60 °C for 2 h, a small peak of preQ₀-Lys nucleoside appeared (Fig. 4B lower). The standard marker (Fig. 4B middle) was prepared by nuclease P1 and bacterial alkaline phosphatase double digestions of tRNA^Phe-preQ₀-Lys. Thus, the minimum substrate for ArcS is preQ₀ nucleoside. Furthermore, when phosphate was attached to the 5′-OH of preQ₀ nucleoside (i. e. preQ₀ nucleoside 5′-monophosphate; 5′P-preQ₀), a clear peak of preQ₀-Lys nucleoside 5′-monophosphate (5′P-preQ₀-Lys) appeared (Fig. 4C lower). When preQ₀ nucleoside was used as a substrate, a very small preQ₀-Lys peak appeared after 2 h incubation (Fig. 4B). In contrast, when 5′P-preQ₀ nucleoside was used, almost all 5′P-preQ₀ nucleoside was converted to 5′P-preQ₀-Lys (Fig. 4C). These data show that the presence of 5′-phosphate is required for the efficient lysine-transfer reaction. Furthermore, when phosphate was attached to the 3′-OH of preQ₀ nucleoside (preQ₀ nucleoside 3′-monophosphate; 3′P-preQ₀), the formation of lysine-adduct (3′P-preQ₀-Lys) by ArcS was not increased (Fig. 4D). Thus, 3′-phosphate is not involved in the substrate recognition by ArcS. Taking these experimental results together, we conclude that the minimum substrate for ArcS is preQ₀ nucleoside and that the presence of 5′-phosphate is required for the efficient lysine-transfer reaction of ArcS.

**preQ**₀**nucleoside and its derivatives are the substrates of ArcS.** The structures of preQ₀ and its derivatives are illustrated in each panel. Asterisks mark a derivative from 2-mercaptoethanol. This derivative is non-enzymatically synthesized during incubation at 60 °C for 2 h. A, ArcS does not transfer lysine to preQ₀ base. B, ArcS transfers lysine to preQ₀ nucleoside very slowly. C, the presence of 5′-phosphate in 5′P-preQ₀ nucleoside accelerates the lysine-transfer reaction of ArcS. D, the presence of 3′-phosphate in 3′P-preQ₀ nucleoside does not accelerate the lysine-transfer reaction of ArcS.

Kinetic study of the lysine-transfer reaction to 5′P-preQ₀-Lys mediated by ArcS

Because the substrate is a nucleotide (or nucleoside), measurement of the velocity of lysine-adduct formation by conventional filter assay with ¹⁴C-lysine is difficult. Therefore, we tested whether the kinetic parameters of ArcS for 5′P-preQ₀ could be measured spectroscopically. Initially, we measured the ultra-violet light absorption spectra of 5′P-preQ₀ (Fig. 5A blue) and 5′P-preQ₀-Lys (Fig. 5A red): these compounds were prepared by organic synthesis. As shown in Figure 5A, 5′ P-preQ₀ did not absorb UV_{320 nm}. In contrast, 5′P-preQ₀-Lys absorbs UV_320nm. Therefore, we hypothesized that the velocity of 5′P-preQ₀-Lys formation could be measured by an increase in absorbance at 320 nm. To verify this idea, we mixed 0.11 μM ArcS-RaSEA complex, 50 μM 5′ P-preQ₀ and 200 μM lysine and then monitored the change of absorbance at 320 nm at 60 °C (Fig. 5B). It should be mentioned that 2-mercaptoethanol was not added into the reaction mixture because 2-mercaptoethanol is converted to a derivative, which absorbs UV_{320 nm} (this derivative is marked by asterisks in Fig. 4). As shown in Figure 5B, the initial velocity of 5′ preQ₀-Lys formation could be measured. We determined the kinetic parameters of ArcS for 5′P-preQ₀ using this method (Fig. 5C and Table 1). The Km value for 5′P-preQ₀ was very large (433.4 μM). However, the Kcat value for 5′P-preQ₀ was comparable to that for a 21 nt or 64 nt RNA fragment. Because the Kcat values for 5′P-preQ₀ and RNA fragments are higher than that for full-length tRNA transcript (Table 1), we propose that the L-shaped tRNA structure may preturb the lysine-transfer reaction mediated by ArcS.

**Measurement of kinetic parameters of ArcS for 5′P-preQ**₀.A, UV-absorption spectra of 5′P-preQ₀-Lys (*red*) and 5′P-preQ₀ (*blue*) at 60 °C. B, the initial velocity of formation of 5′P-preQ₀-Lys was monitored by absorbance at 320 nm. C, kinetic parameters of ArcS for 5′P-preQ0 nucleoside were determined spectroscopically.

The combination of ArcTGT and ArcS and RaSEA complex can form preQ₀-Lys in all tRNA transcripts from T. kodakarensis

ArcS can act on preQ₀ nucleoside. This result suggests that the tRNA specificity of preQ₀-Lys15 formation by ArcS may be determined by the substrate tRNA specificity of ArcTGT. In other words, if ArcTGT introduces a preQ₀ base into tRNA, the preQ₀ may be modified to preQ₀-Lys by ArcS irrespective of the sequences of the tRNAs. As described in the previous section, ArcTGT from P. horikoshii does not recognize the L-shaped tRNA structure and nucleotide sequences (except for G15) in the D-loop but rather recognizes the ribose-phosphate backbone of the D-arm (28). If T. kodakarensis ArcTGT possesses the same tRNA specificity, the combination of ArcTGT and ArcS and RaSEA complex would modify all tRNA transcripts from T. kodakarensis. In the T. kodakarensis genome, 46 tRNA genes are encoded (44). To confirm the above idea, we prepared 46 tRNA transcripts using T7 RNA polymerase (45) and tested whether ArcTGT and ArcS and RaSEA complex form preQ₀-Lys in these tRNA transcripts (Figs. 6 and S3). In this experiment, S. cerevisiae tRNA^Phe transcript was used as a control. As shown in Fig. 6, ¹⁴C-lysine was incorporated into all tRNA transcripts tested. Thus, T. kodakarensis ArcTGT and ArcS and RaSEA complex act on all tRNA species in vitro. However, the velocities of preQ₀-Lys formation differed according to the tRNA species. For example, preQ₀-Lys formation into tRNA^LeuCAG transcript was clearly slower than that into other tRNA transcripts (Fig. 6). In all tRNA species from T. kodakarensis, the presence of G⁺15 has only been confirmed in tRNA^Trp (18). Therefore, we prepared tRNA^LeuCAG and tRNA^Trp transcripts (Fig. 7A) and tested the ¹⁴C-guanine exchange reactions of ArcTGT (Fig. 7B left). There was no significant difference between the velocities of the ¹⁴C-guanine exchange reaction for these tRNA transcripts (Fig. 7B left). In contrast, the velocity of ¹⁴C-lysine-transfer to preQ₀-tRNA^Trp mediated by ArcS (red) was faster than that to preQ₀-tRNA^LeuCAG (blue) (Fig. 7B right). Thus, the slow preQ₀-Lys formation for tRNA^LeuCAG transcript is mainly caused by a slow lysine-transfer by ArcS. This result shows that velocities of lysine-transfer by ArcS differ according to tRNA species although ArcS can act on preQ₀ nucleoside. Moreover, ArcTGT changes the structure of substrate tRNA from the L-shaped structure to the so-called the λ-form (30). Therefore, when a combination of ArcTGT and ArcS and RaSEA complex was used (Fig. 6), the λ-form structure formed by ArcTGT might affect the formation of preQ₀-Lys by the ArcS and RaSEA complex.

**Combination of ArcTGT and ArcS and RaSEA complex from *T. kodakarensis* transfers**¹⁴**C-labeled lysine to all tRNA transcripts tested.** In the *T. kodakarensis* genome, 46 tRNA genes are present. 46 tRNA transcripts were prepared and then tested to determine whether a combination of ArcTGT and ArcS and RaSEA complex transferred ¹⁴C-labeled lysine into them. After the reaction, 0.01 A260 units of tRNAs were loaded onto the gels. The RNAs were visualized by methylene blue staining and autoradiograms of the gels were obtained. *Saccharomyces cerevisiae* tRNA^Phe was used as a control in each gel. Abbreviations are as follows: iMet, initiator tRNA^Met transcript; Met, elongator tRNA^Met transcript.

**The slow preQ**₀**-Lys formation in tRNA**^LeuCAG is caused by slow lysine-transfer of ArcS.A, cloverleaf structures of tRNA^LeuCAG and tRNA^Trp transcripts are shown. B, ¹⁴C-guanine incorporations into tRNA^LeuCAG (*blue*) and tRNA^Trp (*red*) transcripts by ArcTGT are compared (*left*). ¹⁴C-lysine incorporations into tRNA^LeuCAG-preQ₀ (*blue*) and tRNA^Trp-preQ₀ (*red*) transcripts by ArcS are compared. The data were obtained from three independent experiments. Error bars show the standard deviations.

T. kodakarensis ArcS can transfer lysine to mesophilic archaeal tRNAs, which do not possess the G⁺15 modification in cells

Several tRNAs from mesophilic archaea do not possess the G⁺15 modification. For example, in the case of H. volcanii, tRNA^AspGUC, tRNA^AlaCGC and tRNA^ArgGCG possess unmodified G15 (8). Because the set of T. kodakarensis ArcTGT and ArcS and RaSEA complex acts on all tRNA species, we tested whether this set forms preQ₀-Lys in H. volcanii tRNA^AspGUC, tRNA^AlaCGC and tRNA^ArgGCG transcripts. As shown in Figure 8, ¹⁴C-lysine was transferred to H. volcanii tRNA^AspGUC, tRNA^AlaCGC and tRNA^ArgGCG transcripts. Thus, this result suggests that thermophilic archaea G⁺ formation system may possess broader substrate tRNA specificity than the mesophilic archaea system.

**Combination of ArcTGT and ArcS and RaSEA complex from *T. kodakarensis* transfers**¹⁴**C-labeled lysine to *H. volcanii* tRNA transcripts, which do not possess G**⁺**modification in living cells.** Native *H. volcanii* tRNA^Ala, tRNA^Asp and tRNA^Arg do not possess the G⁺15 modification. The combination of ARcTGT and ArcS and RaSEA complex from *T. kodakarensis* transfers ¹⁴C-lysine to these tRNA transcripts. *Saccharomyces cerevisiae* tRNA^Phe was used as a positive control. The RNAs were visualized by methylene blue staining (*left*) and an autoradiogram of the gel was obtained (*right*).

The lysine transfer reaction mediated by ArcS prevents the reverse reaction in G⁺ formation in tRNA

ArcTGT can exchange the preQ₀ base in tRNA with guanine base (25). This reverse reaction perturbs the formation of G⁺ in tRNA. If the intermediate (preQ₀-Lys) prevents the reverse reaction, the lysine-transfer reaction mediated by ArcS would play a role in promoting the anterograde reaction (G⁺ formation). Therefore, we investigated whether ArcTGT can exchange the preQ₀-Lys in tRNA with guanine or preQ₀ base (Fig. 9). As a substrate, S. cerevisiae tRNA^Phe-preQ₀ was near-completely modified to tRNA^Phe-preQ₀-Lys with ¹⁴C-lysine. This tRNA^Phe-preQ₀-¹⁴C-Lys (8 μM) was incubated with buffer at 60 °C for 4 h (control; left lane in Fig. 9). Addition of 1.0 μM ArcTGT and 200 μM guanine base (middle lane) or 200 μM preQ₀ base (right lane) did not change the intensities of tRNA^Phe-preQ₀-¹⁴C-Lys in the autoradiogram. This result shows that ArcTGT does not catalyze the reverse reaction from tRNA-preQ₀-Lys to tRNA-guanine or tRNA-preQ₀. Furthermore, ArcS does not catalyze the reverse reaction or lysine-exchange reaction of preQ₀-Lys in tRNA. To demonstrate this, we performed one experiment (Fig. S4). The tRNA^Phe-preQ₀-¹⁴C-Lys (8 μM) was incubated with buffer at 60 °C for 4 h (control; left lane in Fig. S4). Addition of 0.11 μM ArcS and RaSEA complex (middle lane) and 0.11 μM ArcS and RaSEA complex and 200 μM lysine (right lane) did not change the band intensities of the autoradiogram. These experimental results are consistent with the result in Figure 4: 5′P-preQ₀ nucleoside was near-completely converted to 5′P-preQ₀-Lys, showing that the reverse reaction is not catalyzed by ArcS. Taking all experimental results together, we conclude that the formation of tRNA-preQ₀-Lys by ArcS plays a role in preventing the reverse reaction in G⁺ biosynthesis.

**ArcTGT does not exchange the preQ**₀**-Lys in tRNA by guanine or preQ**₀**base.** G15 in *S. cerevisiae* tRNA^Phe transcript was near-completely modified to preQ₀-¹⁴C-Lys by the combination of ArcTGT and ArcS and RaSEA complex from *T. kodakarensis*. This tRNA transcript (0.1 A260 units each) was incubated in the buffer without proteins (*left*), with 0.5 μM ArcTGT and 200 μM guanine base (*middle*), and with 0.5 μM ArcTGT and 200 μM preQ₀ base (*right*) at 60 °C for 2 h and then analyzed by 10% PAGE (7 M urea) (*left panel*). The RNAs were visualized by methylene blue staining. The band intensities in the autoradiogram (*right panel*) do not differ, demonstrating that ArcTGT does not exchange the preQ₀-Lys in tRNA with guanine or preQ₀ base.

Discussion

In this study, we have focused on the substrate RNA recognition mechanism of ArcS. To our surprise, ArcS can act on preQ₀ nucleoside and the presence of 5′-phosphate accelerates the lysine-transfer reaction. There are several tRNA modification enzymes, which have a broad substrate RNA specificity. For example, S. cerevisiae Trm4 (tRNA 5-methylcytosine methyltransferase) acts at multiple positions in tRNAs (46, 47, 48). Furthermore, the eukaryotic pseudouridine synthase, Pus7 acts at multiple positions in several RNA including tRNA (49, 50, 51). Moreover, archaeal NAT10 forms N4-acetylcytosine at multiple positions in several RNAs including tRNA (52). In addition, several tRNA modification enzymes can act on small RNA fragments such as microhelix RNAs (53, 54, 55, 56, 57, 58). However, these tRNA modification enzymes do not act on nucleoside or nucleotide. To our knowledge, ArcS is an exceptional tRNA modification enzyme that is able to act on nucleoside or nucleotide. In living cells, preQ₀ nucleoside and preQ₀ nucleotide are only produced by the degradation of tRNA-preQ₀15, an intermediate of tRNA-G⁺15 formation. Because the Km value of ArcS for 5′P-preQ₀ nucleoside is very large (433.4 μM), the lysine-transfer reaction to nucleoside or nucleotide by ArcS rarely occurs in cells.

ArcTGT from thermophilic archaea recognizes the ribose-phosphate backbone of the D-arm structure in tRNA (28). Although the loop and stem sizes of D-arm in archaeal tRNAs differ, several nucleosides (A13, pyrimidine17, G18, G19 and A21) including the modification site (G15) are highly conserved. Therefore, we considered whether the combination of ArcTGT and ArcS and RaSEA complex from T. kodakarensis might act on all tRNAs. In fact, preQ₀ in all tRNA transcripts tested was modified to preQ₀-Lys by ArcS in vitro. Furthermore, H. volcanii tRNAs, in which G15 is not modified to G⁺15 in living H. volcanii cells, were also modified by the ArcTGT and ArcS and RaSEA complex from T. kodakarensis. However, the velocities of formation of preQ₀-Lys in tRNA differed between the tRNA transcripts (Fig. 6). For example, the velocity of formation of preQ₀-Lys in the tRNA^LeuCAG transcript was considerably slower compared to that in other tRNA transcripts. This phenomenon was caused by a slow lysine-transfer reaction to preQ₀-tRNA^LeuCAG transcript (Fig. 7). Thus, although ArcS can act on preQ₀ nucleoside, the structure of substrate tRNA affects the lysine-transfer reaction mediated by ArcS. The presence of 5′-phosphate at the modification site (preQ₀15) accelerates the lysine-transfer reaction, suggesting that ArcS captures this phosphate during the lysine-transfer reaction. In the complex between ArcTGT and tRNA, ArcTGT does not capture the 5′-phosphate at the modification site (G15) (30). These observations suggest a difference between the tRNA-binding modes of ArcS and ArcTGT although ArcS and ArcTGT share amino acid sequence homology (34). To clarify this in detail, structural studies of the complex of ArcS, RaSEA, and tRNA are necessary.

Until now, G⁺ has been found only in tRNAs (1). However, our experimental results suggest that the combination of ArcTGT and the ArcS and RaSEA complex may act on other RNAs except for tRNAs if the D-arm-like structure is contained in the RNA.

In general, the modification levels of tRNA in living cells depend on the quantitative balance between substrate tRNA and tRNA modification enzymes. For example, over-expression of Trm10 [tRNA methyltransferase for 1-methylguanosine at position 9 (m¹G9); ref. (59)] in S. cerevisiae cells results in the m¹G9 modification in tRNA species that are ordinarily unmodified in vivo (60). Similarly, although native tRNA^Phe from E. coli does not possess 2′-O-methylguanosine at position 18 (Gm18) (61), E. coli TrmH (tRNA methyltransferase for Gm18 modification; ref. (62, 63)) can methylate tRNA^Phe transcript very slowly under in vitro conditions (64). Therefore, some native tRNA species such as tRNA^LeuCAG may possess unmodified G15 instead of G⁺15. This idea is in line with the observation that elongator tRNA^Met from P. furiosus possesses unmodified G15 (11), although P. furiosus is a hyper-thermophilic archaeon. In addition, it should be mentioned that a mesophilic archaeon, H. volcanii, may have a different G⁺15 modification system. Watanabe et al. purified native ArcTGT from H. volcanii cells (25). This ArcTGT efficiently catalyzes the ¹⁴C-guanine exchange reaction to H. volcanii tRNA^Lys transcript but not to S. cerevisiae and bovine tRNA mixtures (25). Therefore, ArcTGT from H. volcanii may select specific tRNAs as substrates.

In this study, we have clarified the substrate RNA specificity of ArcS. Furthermore, we have established a method for the preparation of tRNA-preQ₀-Lys15. As shown in Figure 4B, preQ₀-Lys was near-completely introduced into tRNA transcript. In fact, in this study, tRNA-preQ₀-Lys15 was used for the experiments described in Figure 9 and Fig. S4. ArcTGT does not exchange the preQ₀-Lys15 in tRNA with guanine or preQ₀ base. Therefore, the formation of preQ₀-Lys15 in tRNA prevents the reverse reaction in G⁺ biosynthesis pathway. In future studies, tRNA-preQ₀-Lys15 will be useful for the analysis of the radical SAM reaction by RaSEA. Moreover, crystal structure studies may be possible using tRNA-preQ₀-Lys15.

Experimental procedures

Materials

L-[¹⁴C(U)]-lysine (11,795.6 MBq/mmol) was purchased from PerkinElmer. Non-radioisotope-labeled lysine (L-lysine monohydrochloride) was obtained from Nacalai Tesque. [8-¹⁴C]-guanine hydrochloride (2123.8 MBq/mmol) was purchased from Moravek. DNA oligomers were obtained from Thermo Fisher Scientific. T7 RNA polymerase was purchased from Toyobo. All other chemical reagents were of analytical grade.

Purification of T. kodakarensis ArcTGT

Purification of T. kodakarensis ArcTGT was described in our previous report (16).

Purification of T. kodakarensis ArcS and RaSEA complex

Purification of T. kodakarensis ArcS and RaSEA complex was described in our previous report (19). In this study, the purification was performed under aerobic conditions.

Preparation of tRNA transcripts

All tRNA transcripts were synthesized using T7 RNA polymerase as described previously (65). DNA oligomers used for the constructions of templates were designed using a Python tool, ROCKET, which was developed in our laboratory (45). The source code for ROCKET is available at Github (https://github.com/TEPPEI-MAT/ROCKET). The sequences of DNA oligomers are listed in Table S1. Two tRNA^Gln and initiator tRNA^Met from T. kodakarensis do not possess G nucleotide at position 1. Therefore, these tRNAs were prepared as precursor forms, which have an attached 5′-leader sequence, which was removed by RNase P digestion as described in a previous report (66). In Figure 6, tRNA^GlnCUG RNase P, tRNA^GlnUUG RNase P, and tRNA^MetCAU RNase P were prepared by the method using RNase P. The purification procedure of RNase P is described in the reference (66). Transfer RNA transcripts were purified by 10% PAGE (7 M urea).

Base, nucleoside, and nucleotide

The guanine base was purchased from Sigma. preQ₀ base was chemically synthesized according to the reference (67). preQ₀ nucleoside 5′-phosphate was synthesized as a triethylammonium salt by 5′-phosphorylation (68) of preQ₀ nucleoside (69). ¹H NMR (400 MHz, D₂O) δ 7.85 (s, 1H), 5.98 (d, J = 6.8 Hz, 1H), 4.57 (dd, J = 6.8, 5.2 Hz, 1H), 4.37 (dd, J = 5.2, 2.9 Hz, 1H), 4.22 (q, J = 2.9 Hz, 1H), 3.93–3.84 (m, 2H), 3.07 (q, J = 7.3 Hz, 12H), 1.18 (t, J = 7.3 Hz, 18H). ³¹P NMR (161.7 MHz, D₂O) δ 4.6 (s, 1P). HRMS (ESI-TOF) m/z: [M − H]⁻ calculated for C₁₂H₁₃N₅O₈P^– 386.0507; found 386.0513. The synthesis of 5′P-preQ₀-Lys nucleoside was described in our previous report (19). Standard markers of preQ₀-Lys nucleoside, 5′P-preQ₀ nucleoside, and 3′P-preQ₀ nucleoside in Figure 4 were prepared by nuclease digestions of tRNA^Phe-preQ₀-Lys. Procedures for preparations of these markers are described in the latter section “Lysine-transfer reaction to preQ0 base, preQ0 nucleoside and preQ0 nucleotides”.

Preparation of S. cerevisiae tRNA^Phe-preQ₀

50.0 A260 units of S. cerevisiae tRNA^Phe transcript, 1 μM ArcTGT, and 200 μM preQ₀ base were incubated in 6.25 ml of buffer A [50 mM Tris-HCl (pH 7.6), 5 mM MgCl₂, 6 mM 2-mercaptoethanol, 50 mM KCl] at 60 °C for 2 h. After the reaction, RNA was treated with phenol-chloroform and recovered by ethanol precipitation.

Measurements of kinetic parameters of ArcS for lysine, tRNA^Phe-preQ_0, and RNA fragments

For measurement of kinetic parameters for lysine, 0.23 μM ArcS and RaSEA complex, 204 μM tRNA^Phe-preQ₀, and various concentrations of ¹⁴C-labeled lysine were incubated in 10 μl of buffer A at 60 °C for 5 min and spotted onto a Whatman 3 MM filter. Incorporations of ¹⁴C-lysine were monitored by conventional filter assay. In brief, the filters were washed in 50 ml 5% trichloroacetic acid solution five times. The filters were dipped into 50 ml 99.5% ethanol to remove water and then dried. The ¹⁴C-lysine incorporation was measured by a liquid scintillation counter. For measurement of kinetic parameters for tRNA-preQ₀ or RNA fragment, 0.11 μM ArcS and RaSEA complex, 200 μM ¹⁴C-lysine, and various concentrations of tRNA^Phe-preQ₀ (or RNA fragment) were incubated in 10 μl of buffer A at 60 °C for 5 min. The aliquot was spotted onto a Whatman 3 MM filter and then incorporations of ¹⁴C-lysine were monitored by the filter assay described above. The data were obtained from three independent experiments. Km and Kcat values were calculated using a Python program developed in our laboratory. This program fits the data to the Michaelis-Menten equation. Error bars show the standard deviations. The source code of the program is described in Supporting Information.

Cleavage of tRNA^Phe-preQ₀ by DNAzymes

In this study, two 8-17 DNAzymes were designed. The sequences are as follows: 8-17 DNAzyme for cleavage between A21 and G22, 5′- CTT CAG TCT GGC GCT TCC GAG CCG GAC GAC CCA ACT GAG CTA AA -3’; 8-17 DNAzyme for cleavage between A64 and G65, 5′- TGG TGC GAA TTT CCG AGC CGG ACG AGT GGA TCG AAC ACA GGA -3’. 0.2 A260 units tRNA^Phe-preQ₀ (final concentration 17 μM), and 25 μM 8-17 DNAzyme was mixed in 20 μl of DNAzyme buffer [20 mM Tris-HCl (pH 7.6), 50 mM MgCl₂, 50 mM NaCl]. The cleavage was performed in a Gene Atlas Thermal Cycler model 482 (ASTEC). The sample was heated at 80 °C for 1 min, gradually cooled to 37 °C over 430 s, and incubated at 37 °C for 30 min. This cycle was repeated ten times and then incubated at 80 °C for 2 min. The RNA fragments were purified by 10% PAGE (7 M urea).

Lysine-transfer reaction to RNA fragments containing preQ₀

0.11 μM ArcS and RaSEA complex, 8.5 μM RNA fragment, and 100 μM ¹⁴C-lysine were incubated in 20 μl of buffer A at 60 °C for 10 min. RNA was treated with phenol-chloroform and recovered by ethanol precipitation. 0.03 A260 units each of RNA was analyzed by 10% PAGE (7 M urea). The gel was stained with 0.2% methylene blue and then dried. The incorporation of ¹⁴C-lysine into RNA was monitored using a Typhoon FLA 7000 system (Cytiva). The kinetic parameters for RNA fragments containing preQ₀ were described in the “Measurements of kinetic parameters of ArcS for lysine, tRNAPhe-preQ0, and RNA fragments” section.

Lysine-transfer reaction to preQ₀ base, preQ₀ nucleoside and preQ₀ nucleotides

Base, nucleoside, and nucleotide analyses were performed with a Hitachi L-2200 HPLC system (Hitachi) equipped with a Nucleosil 7C18 column (4.6 × 250 mm; ChemcoPlus Scientific). The solvent system was previously reported (70). The proteins in the samples were removed using a Durapore Centrifugal Filter Device (PVDF, 0.22 mm; Merck Millipore) and then the flow-through fraction was analyzed. For preQ₀ base analysis (Fig. 4A), 0.23 μM ArcS and RaSEA complex, 17 μM preQ₀ base and 200 μM lysine were incubated in 20 μl of buffer A at 60 °C for 2 h. For preQ₀ nucleoside analysis, S. cerevisiae tRNA^Phe-preQ₀ (0.2 A260 units) was digested with 2.5 units nuclease P1 (Fuji Film-Wako) in 18 μl 50 mM sodium acetate (pH 5.3) at 37 °C for 2 h. After the nuclease P1 digestion, 0.5 units bacterial alkaline phosphatase and 1 μl 1 M Tris-HCl (pH 8.0) were added into the sample and then incubated at 37 °C overnight. This sample is used as “Before reaction” in Figure 4B. In the case of “Standard” in Figure 4B, 0.23 μM ArcS and RaSEA complex, 0.2 A260 units S. cerevisiae tRNA^Phe-preQ₀ and 200 μM lysine were incubated in 20 μl of buffer A at 60 °C for 2 h. The RNA was treated with phenol-chloroform and recovered by ethanol precipitation. The RNA was digested with nuclease P1 and alkaline phosphatase as described above. In the case of “After reaction”, 0.2 A260 units S. cerevisiae tRNA^Phe-preQ₀ was digested to nucleosides with nuclease P1 and alkaline phosphatase as described above. 0.23 μM ArcS and RaSEA complex and 10 x buffer A were added into the sample and then incubated at 60 °C for 2 h. For 5′P-preQ₀ nucleoside analysis (Fig. 4C), S. cerevisiae tRNA^Phe-preQ₀ (0.2 A260 units) was digested with 2.5 units nuclease P1 in 18 μl 50 mM sodium acetate (pH 5.3) at 37 °C for 2 h. This sample was used for “Before reaction” in Figure 4C. In the cases of “Standard” and “Before reaction” in Figure 4C, the samples were not treated with bacterial alkaline phosphatase. For 3′P-preQ₀ nucleoside analysis (Fig. 4D), S. cerevisiae tRNA^Phe-preQ₀ (0.2 A260 units) was digested with 5 units RNase T2 (Sankyo) in 20 μl ammonium acetate (pH 5.0). The 3′phosphate-nucleosides from tRNA were analyzed using the same method as described above.

Spectroscopic analysis of lysine-transfer reaction

UV absorption spectra of 50 μM 5′P-preQ₀ and 50 μM 5′P-preQ₀-Lys in buffer B [50 mM Tris-HCl (pH 7.6), 5 mM MgCl₂, 50 mM KCl] were recorded at 60 °C using an Ultrospec 6300 pro photometer (GE Healthcare). The initial velocity of the lysine-transfer reaction to 5′P-preQ₀ in Figure 5B was determined as follows. 100 μM 5′P-preQ₀ and 200 μM lysine in 400 μl buffer B were prewarmed at 60 °C and the reaction was started by the addition of ArcS and RaSEA complex (final concentration is 0.11 μM). The kinetic parameters were calculated from the velocities of lysine-transfer to various concentrations of 5′P-preQ₀.

Measurements of ¹⁴C-lysine-incorporations into tRNA transcripts

1.0 μM ArcTGT, 0.23 μM ArcS and RaSEA complex, 0.1 A260 units tRNA transcript, 500 μM preQ_0, and 100 μM lysine were incubated in 20 μl buffer A at 60 °C for 5 min. To remove K⁺ ions, the buffer A in the samples was exchanged with water using an Amicon Ultra Centrifugal Filter device (Millipore, 10,000 MWCO, code number UFC501024). RNAs were treated with phenol-chloroform and recovered by ethanol precipitation. 0.01 A260 units RNAs were analyzed by 10% PAGE (7 M urea). The gels were stained with 0.2% methylene blue and then dried. Autoradiograms of the gels were obtained using a Typhoon FLA 7000 system (Cytiva).

Analysis of base exchange reaction by ArcTGT

0.30 A260 units S. cerevisiae tRNA^Phe-preQ₀, 0.23 μM ArcS and RaSEA complex and 300 μM ¹⁴C-lysine were incubated in 30 μl buffer A at 60 °C for 2 h. RNA was treated with phenol-chloroform and then recovered by ethanol precipitation. The resultant tRNA^Phe-preQ₀-¹⁴C-Lys was dissolved in 20 μl water. 0.10 A260 units tRNA^Phe-preQ₀-¹⁴C-Lys, 0.5 μM ArcTGT and 200 μM guanine base (or preQ₀ base) were incubated in 20 μl buffer A at 60 °C for 2 h. 0.03 A260 units RNAs were analyzed by 10% PAGE (7 M urea). The gels were stained with 0.2% methylene blue and then dried. Autoradiogram of the gel was obtained using a Typhoon FLA 7000 system (Cytiva). For the experiment in Figure 7B left, 0.10 A260 units T. kodakarensis tRNA^LeuCAG (or tRNA^Trp), 85 μM ¹⁴C-guanine, and 0.75 μM ArcTGT were incubated in 20 μl buffer A at 60 °C for 0, 2, 5, 10, and 20 min. The incorporation of ¹⁴C-guanine was monitored by filter assay as described in the previous section. For the experiment in Figure 7B right, 0.10 A260units T. kodakarensis tRNA^LeuCAG-preQ₀ (or tRNA^Trp-preQ₀), 300 μM ¹⁴C-lysine and 0.11 μM ArcS and RaSEA complex were incubated in 20 μl buffer A at 60 °C for 0, 1, 2, 5, 10, 15, and 20 min. The incorporation of ¹⁴C-lysine was monitored by filter assay as described in the previous section.

Data availability

All data used in this study are available from the corresponding author (H. H.) upon request.

Supporting information

This article contains supporting information.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Acknowledgments

We thank Dr Yukihide Ishibashi and Dr Tsuyoshi Asahi (Ehime University) for technical support in the preliminary experiments of spectroscopic assay, Mr Teppei Matsuda (Ehime University) for assistance of use of a Python software, ROCKET, Mr Keita Hiura (Gifu University) for assistance of expression of recombinant proteins in E. coli cells, and the RI room staffs in the Advanced Research Support Center (Ehime University) for use of radioisotope compounds.

Author contributions

T. Y., H. H., A. H. supervision, T. Y., T. K., N. O., R. Y., Y. S., and S. F. investigation. H. H. and S. F. writing–original draft, H. H., A. H. funding acquisition; S. F. data curation.

Funding and additional information

This work was partly supported by Grant-in-Aid for Scientific Research from the JAPAN Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP20H03211 and JP24K09381 (to H. H.) and Funding from the Institute for Fermentation, Osaka, Grant Number G-2022-2-052 (to A. H.).

Reviewed by members of the JBC Editorial Board. Edited by Karin Musier-Forsyth

Footnotes

Present address for Takuya Kawamura: Computational Medicine Center, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

Supporting information

Supporting Table

mmc1.xlsx^{(13.5KB, xlsx)}

Supporting Information

mmc2.pdf^{(396.5KB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Table

mmc1.xlsx^{(13.5KB, xlsx)}

Supporting Information

mmc2.pdf^{(396.5KB, pdf)}

Data Availability Statement

All data used in this study are available from the corresponding author (H. H.) upon request.

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PERMALINK

ArcS from Thermococcus kodakarensis transfers L-lysine to preQ0 nucleoside derivatives as minimum substrate RNAs

Shu Fujita

Yuzuru Sugio

Takuya Kawamura

Ryota Yamagami

Natsuhisa Oka

Akira Hirata

Takashi Yokogawa

Hiroyuki Hori

Abstract

Figure 1.

Results

Purification of recombinant ArcTGT, ArcS, and RaSEA complex

Measurement of kinetic parameters of lysine transfer reaction

Figure 2.

Table 1.

ArcS does not require the L-shaped tRNA structure for the lysine-transfer reaction

Figure 3.

ArcS can transfer lysine to preQ0 nucleoside and the presence of 5′-phosphate accelerates the reaction

Figure 4.

Kinetic study of the lysine-transfer reaction to 5′P-preQ0-Lys mediated by ArcS

Figure 5.

The combination of ArcTGT and ArcS and RaSEA complex can form preQ0-Lys in all tRNA transcripts from T. kodakarensis

Figure 6.

Figure 7.

T. kodakarensis ArcS can transfer lysine to mesophilic archaeal tRNAs, which do not possess the G+15 modification in cells

Figure 8.

The lysine transfer reaction mediated by ArcS prevents the reverse reaction in G+ formation in tRNA

Figure 9.

Discussion

Experimental procedures

Materials

Purification of T. kodakarensis ArcTGT

Purification of T. kodakarensis ArcS and RaSEA complex

Preparation of tRNA transcripts

Base, nucleoside, and nucleotide

Preparation of S. cerevisiae tRNAPhe-preQ0

Measurements of kinetic parameters of ArcS for lysine, tRNAPhe-preQ0, and RNA fragments

Cleavage of tRNAPhe-preQ0 by DNAzymes

Lysine-transfer reaction to RNA fragments containing preQ0

Lysine-transfer reaction to preQ0 base, preQ0 nucleoside and preQ0 nucleotides

Spectroscopic analysis of lysine-transfer reaction

Measurements of 14C-lysine-incorporations into tRNA transcripts

Analysis of base exchange reaction by ArcTGT

Data availability

Supporting information

Conflict of interest

Acknowledgments

Author contributions

Funding and additional information

Footnotes

Supporting information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

ArcS from Thermococcus kodakarensis transfers L-lysine to preQ₀ nucleoside derivatives as minimum substrate RNAs

ArcS can transfer lysine to preQ₀ nucleoside and the presence of 5′-phosphate accelerates the reaction

Kinetic study of the lysine-transfer reaction to 5′P-preQ₀-Lys mediated by ArcS

The combination of ArcTGT and ArcS and RaSEA complex can form preQ₀-Lys in all tRNA transcripts from T. kodakarensis

T. kodakarensis ArcS can transfer lysine to mesophilic archaeal tRNAs, which do not possess the G⁺15 modification in cells

The lysine transfer reaction mediated by ArcS prevents the reverse reaction in G⁺ formation in tRNA

Preparation of S. cerevisiae tRNA^Phe-preQ₀

Measurements of kinetic parameters of ArcS for lysine, tRNA^Phe-preQ_0, and RNA fragments

Cleavage of tRNA^Phe-preQ₀ by DNAzymes

Lysine-transfer reaction to RNA fragments containing preQ₀

Lysine-transfer reaction to preQ₀ base, preQ₀ nucleoside and preQ₀ nucleotides

Measurements of ¹⁴C-lysine-incorporations into tRNA transcripts