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Consumption of N5, N10-methylenetetrahydrofolate in Thermus thermophilus under nutrient-poor condition

Consumption of N5, N10-methylenetetrahydrofolate in Thermus thermophilus under nutrient-poor... Abstract TrmFO catalyzes the formation of 5-methyluridine at position 54 in tRNA and uses N5, N10-methylenetetrahydrofolate (CH2THF) as the methyl group donor. We found that the trmFO gene-disruptant strain of Thermus thermophilus, an extremely thermophilic eubacterium, can grow faster than the wild-type strain in the synthetic medium at 70°C (optimal growth temperature). Nucleoside analysis revealed that the majority of modifications were appropriately introduced into tRNA, showing that the limited nutrients are preferentially consumed in the tRNA modification systems. CH2THF is consumed not only for tRNA methylation by TrmFO but also for dTMP synthesis by ThyX and methionine synthesis by multiple steps including MetF reaction. In vivo experiment revealed that methylene group derived from serine was rapidly incorporated into DNA in the absence of TrmFO. Furthermore, the addition of thymidine to the medium accelerated growth speed of the wild-type strain. Moreover, in vitro experiments showed that TrmFO interfered with ThyX through consumption of CH2THF. Addition of methionine to the medium accelerated growth speed of wild-type strain and the activity of TrmFO was disturbed by MetF. Thus, the consumption of CH2THF by TrmFO has a negative effect on dTMP and methionine syntheses and results in the slow growth under a nutrient-poor condition. DNA synthesis, MetF, RNA modification, ThyX, TrmFO To date more than 90 modified nucleosides have been found in tRNA (1) and biosynthesis pathways of these modified nucleosides often include a methylation step(s) by a tRNA methyltransferase(s) (2). In general, tRNA methyltransferases consume S-adenosyl-l-methionine (AdoMet) as the methyl group donor. However, exceptionally, two eubacterial tRNA modification enzymes, TrmFO (3) and the MnmE (4, 5) and MnmG complex (MnmEG) (6, 7), use N5, N10-methylenetetrahydrofolate (CH2THF) as the methyl group donor (Supplementary Fig. S1A). The trmFO genes are present in the genomes of almost all gram-positive and 70% of gram-negative bacteria (3) whereas the mnmE and mnmG genes have been found widely in eubacterial genomes (8). Although both TrmFO and MnmEG are CH2THF- and FAD-dependent tRNA modification enzymes, these enzymes modify uridines at different positions in tRNA: TrmFO is required for the formation of 5-methyluridine at position 54 (m5U54) in the T-loop, whereas MnmEG is needed for the 5-methylaminomethyluridine (mnm5U34) and the 5-carboxymethylaminomethyluridine (cmnm5U34) modifications at position 34 in the anticodon loop (Supplementary Fig. S1B). The m5U54 has been commonly found in tRNAs from eukaryotes, eubacteria and some archaea (2, 9–11) while mnm5U34 and cmnm5U34 derivatives have been found in limited numbers of eubacterial tRNA species: in the case of Escherichia coli, only six tRNA species possess mnm5U34 or cmnm5U34 derivatives (12). Therefore, m5U54 formation is the main pathway among tRNA modification systems in which CH2THF is consumed (Supplementary Fig. S1). It should be mentioned that some gram-negative bacteria including E. coli have an AdoMet-dependent tRNA (m5U54) methyltransferase (TrmA) (13, 14). Therefore, in the case of E. coli, for tRNA methylation, CH2THF is only used by MnmEG. Furthermore, Mycoplasma capricolum exceptionally possesses a folate- and flavin-dependent rRNA methyltransferase (15): this methyltransferase has not been found in the other eubacteria. The m5U54 forms a reverse Hoogsteen base pair with A58 in tRNA (16–18): A58 is often modified to 1-methyladenosine at position 58 (m1A58) (19). The m5U54-A58 (or m1A58) base pair stacks with the G53-C61 base pair in the T-arm and as a result, the L-shaped tRNA structure is stabilized. Therefore, m5U54 is widely found in tRNAs in three domains of life (1, 2, 9). In thermophilic eubacteria such as Thermus thermophilus (20–22) and Aquifex aeolicus (23), and some thermophilic archaea (24), m5U54 is further modified to 5-methyl-2-thiouridine (m5s2U54). The 2-thiomodification at position 54 shifts the equilibrium of ribose puckering to the C3′-endo form and the introduced sulphur atom enhances the stacking effect between the m5U54-A58 (or m1A58) and G53-C61 base pairs (25). In 2009, we determined the X-ray crystal structure of T. thermophilus TrmFO (26). In 2012, we devised an in vitro system to assay TrmFO activity and elucidated the substrate tRNA recognition mechanism of TrmFO: TrmFO recognizes the conserved U54U55C56 sequence and G53-C61 base pair in the T-arm structure (27). The hypothetical catalytic mechanism of the reaction by TrmFO has been proposed by Hamdane et al. (28–30). Thermus thermophilus can grow at wide ranges of temperature (50–83°C) (31). In recent studies (32–36), we reported that modified nucleotides in tRNA and tRNA modification enzymes in T. thermophilus form a network and that this network changes the degrees of modification in tRNAs according to the temperature of the culture. The protein synthesis system in T. thermophilus can adapt to the changes in temperature through alteration of structural flexibility (rigidity) of tRNA (22, 25, 32–34, 36). Figure 1A shows the modifications and responsible enzymes in T. thermophilus tRNAPhe (32, 37). The network at low temperatures (below 55°C) is illustrated in Fig. 1B. For example, at 50°C, the pseudouridine at position 55 (Ψ55) in tRNA prevents excess 2′-O-methylguanosine at position 18 (Gm18) modification by TrmH and m1A58 modification by TrmI (33). When tRNA (Ψ55) synthase gene (truB) was disrupted, the modification level of m5s2U54 in tRNA at 50°C increased (33). Because the m1A58 modification is required for sulphur transfer in the formation of m5s2U54 at 80°C (22), the negative effect of Ψ55 on the m5s2U54 formation at 50°C may be indirectly caused by the effect of m1A58. Totally, the presence of Ψ55 prevents excessive formation of Gm18, m1A58 and m5s2U54 at 50°C (33). Therefore, the Ψ55 modification contributes to maintaining the flexibility of tRNA at low temperatures. In contrast, at 80°C, the modification of G46 by TrmB (to m7G46 (32, 38–40)) accelerates formation of tRNA modifications, Gm18 (by TrmH (41–43)), m1A58 (by TrmI (19, 34)) and m1G37 (by TrmD (44)), which prevent structural disruption of tRNA at high temperatures (32). At low temperatures (below 55°C), the effect of m7G46 on other modifications is unclear (Fig. 1B). Furthermore, we reported that the dihydrouridine at position 20 (D20) formed by Dus did not have an effect on the network (35). Moreover, we investigated the role of m5U54 modification in the network: the absence of m5U54 modification in tRNA causes the increase of Gm18 and slight decrease of m1A58 at 50°C (36). Thus, the m5U54 modification in tRNA coordinately works with the Ψ55 modification to maintain the balance of tRNA modifications at low temperatures (Fig. 1B). The trmFO gene disruptant (ΔtrmFO) strain showed slight growth retardation in the nutrient-rich medium at 50°C (36). Fig. 1 View largeDownload slide Transfer RNA modification network in T. thermophilus at low temperatures. (A) The modified nucleosides and responsible enzymes in T. thermophilus tRNAPhe are illustrated. Bold letters show the modified nucleosides and positions. The responsible enzymes are connected by arrows. Abbreviations of modified nucleosides are as follows; m2G, N2-methylguanosine; s4U, 4-thiouridine; Gm, 2′-O-methylguanosine; D, dihydrouridine; i6A, N6-isopetenyladenosine; ms2i6A, 2-methylthio-N6-isopentenyladenosine; Ψ, pseudouridine; m7G, 7-methylguanosine; m5U, 5-methyluridine; m5s2U, 5-methyl-2-thiouridine; m1A, 1-methyladenosine. (B) The network between the modified nucleosides in tRNA and tRNA modification enzymes in T. thermophilus at low temperatures is illustrated. As described in the main text, this network contributes to maintaining the balance of modification levels in tRNA. Fig. 1 View largeDownload slide Transfer RNA modification network in T. thermophilus at low temperatures. (A) The modified nucleosides and responsible enzymes in T. thermophilus tRNAPhe are illustrated. Bold letters show the modified nucleosides and positions. The responsible enzymes are connected by arrows. Abbreviations of modified nucleosides are as follows; m2G, N2-methylguanosine; s4U, 4-thiouridine; Gm, 2′-O-methylguanosine; D, dihydrouridine; i6A, N6-isopetenyladenosine; ms2i6A, 2-methylthio-N6-isopentenyladenosine; Ψ, pseudouridine; m7G, 7-methylguanosine; m5U, 5-methyluridine; m5s2U, 5-methyl-2-thiouridine; m1A, 1-methyladenosine. (B) The network between the modified nucleosides in tRNA and tRNA modification enzymes in T. thermophilus at low temperatures is illustrated. As described in the main text, this network contributes to maintaining the balance of modification levels in tRNA. During the course of study (36), we found that the ΔtrmFO strain can grow faster than the wild-type strain in the synthetic medium even at 70°C (optimal growth temperature for T. thermophilus) (Fig. 2A). CH2THF is consumed by other metabolic pathways such as dTMP, purine and methionine syntheses (see Fig. 7). CH2THF is the methyl group donor of thymidylate synthase (ThyX), which catalyzes the methylation of dUMP to form dTMP (45, 46). Because CH2THF is labile and the amount of CH2THF supplied by serine hydroxymethyltransferase (SHMT) in living cells is limited, the synthesis of dTMP by ThyX is expected to be the rate-limiting step in DNA synthesis (47). We assumed that the consumption of CH2THF by TrmFO might have an effect on the DNA synthesis and/or other metabolisms of one-carbon via folate derivatives in living cells. In this paper, we report that the consumption of CH2THF by TrmFO under a nutrient-poor condition have a negative effect on the DNA and methionine syntheses. Fig. 2 View largeDownload slide The trmFO gene-disruptant (ΔtrmFO) strain can grow faster than the wild-type strain under the nutrient-poor condition at 70°C. (A) The growth of ΔtrmFO strain (open circles) was faster than that of the wild-type strain (filled circles) in the synthetic minimal medium at 70°C (the data are averages of six independent experiments. (n = 6)). The arrow shows the time point that 3H-serine was added to the medium in the experiment in Fig. 4A. (B) Modified nucleosides in the tRNA fraction from the wild-type strain cells, which were cultured in the nutrient-rich medium at 70°C. Unknown compound (*) from phosphodiesterase, which was used for the digestion of tRNAs, overlapped with the peak of m5U. Furthermore, s2U overlapped with the peak of G. Details were described in our previous paper (36). (C) Modified nucleosides in the tRNA fraction from the wild-type strain cells, which were cultured in the nutrient-poor medium at 70°C. (D) The tRNA fractions from the wild-type (left) and ΔtrmFO (right) strain cells, which were cultured under the nutrient-poor condition, were methylated by E. coli TrmA to estimate the m5U modification levels in tRNA. Fig. 2 View largeDownload slide The trmFO gene-disruptant (ΔtrmFO) strain can grow faster than the wild-type strain under the nutrient-poor condition at 70°C. (A) The growth of ΔtrmFO strain (open circles) was faster than that of the wild-type strain (filled circles) in the synthetic minimal medium at 70°C (the data are averages of six independent experiments. (n = 6)). The arrow shows the time point that 3H-serine was added to the medium in the experiment in Fig. 4A. (B) Modified nucleosides in the tRNA fraction from the wild-type strain cells, which were cultured in the nutrient-rich medium at 70°C. Unknown compound (*) from phosphodiesterase, which was used for the digestion of tRNAs, overlapped with the peak of m5U. Furthermore, s2U overlapped with the peak of G. Details were described in our previous paper (36). (C) Modified nucleosides in the tRNA fraction from the wild-type strain cells, which were cultured in the nutrient-poor medium at 70°C. (D) The tRNA fractions from the wild-type (left) and ΔtrmFO (right) strain cells, which were cultured under the nutrient-poor condition, were methylated by E. coli TrmA to estimate the m5U modification levels in tRNA. Materials and Methods Materials [Methyl-14C]-AdoMet (1.95 GBq/mmol), [methyl-3 H]-AdoMet (2.47 TBq/mmol), l-[3 H(G)]-serine (1.48 TBq/mmol), l-[14C(U)]-serine (1.85 GBq/mmol) and [methyl-3 H]-thymidine (3.33 TBq/mmol) were purchased from Perkin Elmer Life Science. Standard 5-methyluridine (m5U), tetrahydrofolate (THF), adenine and N5-methyltetrahydoxylfolate (5-methylTHF) were obtained from Sigma Aldrich. DE81 filter (code number 3658-323) and 3 MM filter were purchased from Whatman. DNA oligonucleotides were purchased from Invitrogen. HiTrap Heparin HP, HiTrap Blue HP, Hi Trap Q HP and Q-Sepharose columns were purchased from GE Healthcare. The other chemical reagents were of analytical grade. Strain and media The culture source of T. thermophilus HB8 and the procedure for culturing this strain on synthetic minimal medium were a kind gift from Dr Tairo Oshima (Kyowa-kako, Japan). Nutrient-rich medium contained 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, pH 7.5 (adjusted by NaOH) and the medium was supplemented with 0.35 mM CaCl2 and 0.17 mM MgCl2 after autoclaving. The synthetic medium contained the following components (per l litter): 20 g sucrose, 20 g sodium glutamate, 0.5 g K2HPO4, 0.25 g KH2PO4, 2 g NaCl, 0.5 g (NH4)2SO4, 0.1 mg biotin, 1 mg thiamine, 125 mg MgCl2-6H2O, 25 mg CaCl2-2H2O, 1.15 mg NaMoO4-2H2O, 0.1 mg VOSO4-xH2O, 0.5 mg MnCl2-4H2O, 0.06 mg ZnSO4-7H2O, 0.01 mg CuSO4-5H2O, 0.8 mg CoCl2-6H2O, 0.02 mg NiCl2-6H2O, 0.03 µl H2SO4 and 6 mg FeSO4-7H2O. To make plates, gellan gum (Wako Pure Chemicals) was added to the medium (final concentration, 1.5%). Preparation of tRNA mixtures and nucleoside analysis Total RNA was prepared as described in our recent report (48). The tRNA fraction was further purified by 10% polyacrylamide gel containing 7 M urea electrophoresis [PAGE (7 M urea)]. Nucleoside analysis was performed after complete digestion of tRNA with phosphodiesterase, RNase A and bacterial alkaline phosphatase as described previously (32). In vitro methylation of tRNA Transfer RNA mixture (0.20 A260units) from the wild-type or ΔtrmFO strain was methylated by 5 μg of TrmA and 30 μM of 14C-AdoMet at 37°C for 3 h in 50 μl buffer A [50 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 6 mM 2-mercaptoethanol and 50 mM KCl]. The expression and purification of recombinant E. coli TrmA were previously reported (33). The incorporation of the 14C-methyl group was monitored with a liquid scintillation counter (LSC) using a filter assay. Incorporation of radio-labelled serine A total of 370 kBq of 3H-labelled serine was added to 10 ml of culture which was further incubated at 70°C for 4 h. The cells were collected by centrifugation at 6100 × g at 4°C for 2 min, washed with 500 μl of fresh medium and collected. The genomic DNA fraction was extracted from these cells by means of the NucleoSpin Tissue kit (Takara) according to the manufacturer’s instructions and 1 μg of genomic DNA was spotted onto a 3MM filter, and 3H-methyl group incorporation was measured with a LSC. RNase A and DNase I treatments were performed as follows: 400 ng of the genomic DNA was treated with 40 μg of RNase A (Nacalai Tesque) or 20 units of DNase I (Roche) in 30 μl of buffer A at 37°C for 60 min, and the 3H-methyl group content in the sample was measured with a LSC. Expression and purification of recombinant proteins for TrmFO activity measurement The E. coli expression systems and purification procedures used for TrmFO and SHMT were as described previously (26). Expression and purification of ThyX Thermus thermophilus ThyX (TTHA1096)-pET11b expression vector was purchased from RIKEN Biological Resource Center (49) and ThyX was expressed using E. coli BL21 (DE3) Rosetta2 (Novagen) according to the manufacture’s manual. In short: harvested cells (2.8 g) were suspended in 15 ml of buffer A, in which 1 tablet of protease inhibitor cocktail (Roche) was dissolved, and lysed in an ultrasonic disruptor (model VCX-500, Sonics and Materials., Inc., USA) at 4°C. The cell debris was removed by centrifugation at 6000 × g at 4°C for 20 min. The supernatant was heated at 70°C for 20 min, and denatured proteins were removed by centrifugation at 9000 × g at 4°C for 20 min. The supernatant was loaded onto a Hi Trap Q HP column (5 ml) equilibrated with buffer A. ThyX was eluted by a linear gradient of 50 mM–1 M KCl in buffer A. The eluted ThyX fractions were collected, dialyzed against buffer A using a Spectra/Por 7 dialysis membrane (MWCO: 10,000, code 132119, Spectrum Laboratories) at 4°C, and then loaded onto a Hi Trap Blue HP column (5 ml). The bound proteins were eluted by a linear gradient of 50 mM–1.5 M KCl in buffer A. The ThyX fractions were collected and concentrated in a Vivaspin 20-10K ultrafiltration membrane device (GE Healthcare). The concentrated ThyX fraction (3 ml) was loaded onto a HiLoad Superdex 75 preparation-grade gel-filtration column (120 ml, GE Healthcare) equilibrated with buffer A containing 400 mM KCl. The eluted ThyX fractions were collected, dialyzed against buffer A, and concentrated. The purified ThyX (5.7 mg) was mixed with an equivalent volume of 100% glycerol and stocked at −30°C. The purity was checked by 15% SDS-PAGE. Preparation of polyclonal antibodies and western blotting analysis Customized rabbit anti-TrmFO, anti-SHMT and anti-ThyX serums were prepared by Kitayama Labes, Japan, using the purified recombinant proteins as the antigens. Polyclonal antibody fractions were prepared using an Econo-pac serum IgG purification kit (Bio-Rad). Western blotting analysis was performed as described in our recent report (35). Measurement of ThyX activity To visualize the methyl group transfer reaction, we set up a TLC assay system for ThyX. The reaction mixtures (0.4 μM SHMT, 5 μM THF, 5 mM NADPH, 25 μM 14C-labelled serine and 170 μM dUMP in buffer A) were incubated at 60°C and samples were taken at the indicated time points. The methyl transfer reaction was started with the addition of ThyX (final concentration, 0.5 μM). The reaction was stopped by the addition of an equal volume of phenol/chloroform (1:1), and the aqueous phases were recovered. Aliquots (1.5 μl) were spotted onto a thin-layer plate (TLC Cellulose F, code 1.05565.0001, Merck) and the sample was separated by a solvent system of isopropyl alcohol: HCl:H2O, 70:15:15, v/v/v. The 14C-labelled compounds were visualized by Typhoon FLA 7000. The mobility of standard markers was detected by irradiation of UV at 254 nm and by the ninhydrin reaction. Disturbance competition assay between TrmFO and ThyX The reaction mixture containing various concentration of SHMT, 5 μM THF, 5 mM NADPH, 25 μM 3H-labelled serine, 5.6 μM tRNAPhe transcript, and 170 μM dUMP in buffer A was pre-incubated at 60°C for 10 min. The methyl transfer reaction was started by the addition of TrmFO and ThyX, and the reaction mixture was incubated at 60°C for 10 min. Aliquots of 25 μl were spotted onto a DE81 paper. The filter was washed with 250 mM Na2HPO4 for at least five times at room temperature. The methyl group incorporation into tRNAPhe transcript was measured with a LSC. Cultures in synthetic medium with methionine or adenine Methionine was dissolved in water (final concentration, 50 mg/ml), and then pH was adjusted by 5 M NaOH to 7.0. The dissolved methionine was directly added to the synthetic medium. The culture was performed at 70°C. Adenine was directly added to the synthetic medium (final concentration, 100 μM). Expression and purification of MetF Thermus thermophilus MetF (TTHA0327)-pET11b expression vector was purchased from RIKEN Biological Resource Center (49) and MetF was expressed using E. coli Rosetta2 (DE3). Purification of MetF was performed according to the reference (50) with slight modifications. In brief, cells (2.0 g) was suspended in 10 ml of buffer B (50 mM potassium phosphate (pH 7.2), 200 mM NaCl), in which 1 tablet of protease inhibitor cocktail (Roche) was dissolved. The cells were disrupted in an ultrasonic disruptor (model VCX-500, Sonics and Materials, Inc., USA) at 4°C. The cell debris was removed by centrifugation at 20,000 × g at 4°C for 15 min. The supernatant was heated at 70°C for 20 min, and denatured proteins were removed by centrifugation at 8000 x g at 4°C for 20 min. The supernatant was loaded onto a Q-Sepharose column (volume, 5 ml) and then separated by a NaCl linear gradient (200–1000 mM). The eluted sample was dialyzed against buffer A. After dialysis, the precipitant was removed by centrifugation at 8000 × g at 4°C for 20 min. The supernatant fraction (33.2 mg of purified MetF) was concentrated in a Vivaspin 20-10K ultrafiltration membrane device. Activity measurement of MetF NADH oxidase activity of MetF was measured by monitoring of decrease of absorbance at 340 nm at 20°C as follows. 0.4 μM SHMT, 5 μM THF, 200 μM NADH and 25 μM serine in 1 ml of buffer A were pre-incubated at 20°C for 10 min, and then MetF was added (final concentration, 3 μM). Formation of 14C-labelled 5-methylTHF was confirmed by thin-layer chromatography using 14C-labelled serine: To separate CH2THF and 5-methylTHF, the solvent system (phenol-water: conc. ammonia-water, 99) was used. The mobility of 5-methylTHF was checked by UV at 360 nm irradiation using the standard compound. Competitive assay between TrmFO and MetF A total of 0.4 μM SHMT, 5 μM THF, 5 mM NADH, 5 mM NADPH, 1 mM FAD, 25 μM tRNAPhe transcript, 0.75 μM TrmFO, 25 μM 14C-labelled serine, and 0, 0.75, 2.25 or 7.5 μM MetF in 200 μl of buffer A were incubated at 60°C and then 35 μl of aliquot was taken at 0, 10, 20, 30 and 40 min period. Transfer RNA transcripts were recovered by phenol-chloroform treatment and ethanol precipitation, and then analysed by 10% PAGE (7 M urea). The gels were stained with methylene blue and 14C-methyl group incorporations were monitored by Typhoon FLA 7000. Results The ΔtrmFO strain grows faster than the wild-type under nutrient-poor condition at 70°C Under a nutrient-poor condition (minimal synthetic medium), the ΔtrmFO strain grew faster than the wild-type strain at 70°C (Fig. 2A). Because the m5U54 modification in tRNA supports the maintenance of balance of tRNA modifications only at low temperatures (36), this phenomenon at 70°C was not simply explainable by the effect of m5U54 in the network. At the start of this study, we assumed that some modifications are abnormally (or insufficiently) introduced into tRNA under the nutrient-poor condition. To verify this idea, we purified tRNA fractions from the wild-type, which were cultured under nutrient-rich and -poor conditions at 70°C. The modified nucleosides in these tRNA fractions were compared by HPLC C18-reverse-phase column chromatography (Fig. 2B and C). Contrary to our expectation, we could not find any abnormal modifications in the tRNA fraction from the cells cultured under the nutrient-poor condition (Fig. 2B and C): the majority of tRNA modifications (Ψ, m1A, Cm, m7G, m5U, s4U, Gm, m1G, m2G and m5s2U) were sufficiently introduced into the tRNA fraction, showing that the limited nutrients are preferentially consumed in the tRNA modification systems. It should be mentioned that mnm5U derivatives were not detected in Fig. 2B and C because they are hydrophobic and are contained in the limited tRNA species. Furthermore, we checked the modification levels of tRNAs from the ΔtrmFO strain that were cultured in the synthetic minimal medium at 70°C (Supplementary Fig. S2). As shown in Supplementary Fig. S2, we could not find any abnormal modification in this fraction. The peak of m5U overlapped with the peak of unknown compound (asterisks in Fig. 2 and Supplementary Fig. S2) derived from phosphodiesterase, which was used for digestion of tRNA. Furthermore, the s2U and m5s2U peaks overlapped with the peaks of G and m2G, respectively. Therefore, to know the m5U54 and m5s2U54 modification levels, we measured the methylation capacity in the tRNA fractions of wild-type and ΔtrmFO strains by TrmA [E. coli tRNA (m5U54) methyltransferase (13, 14)] and 14C-labelled AdoMet. E. coli TrmA does not catalyze the exchange reaction of methyl group in tRNA (51) and is able to convert s2U54 to m5s2U54 (36). The tRNA fraction from the ΔtrmFO strain did not contain the m5U54 and m5s2U54 modifications (positive control). As shown in Fig. 2D, the 5-carbon in U54 in the tRNA fraction from the wild-type strain cultured under the nutrient-poor condition was near-fully methylated. These experimental results suggested that the rapid growth of ΔtrmFO strain under the nutrient-poor condition was not explainable by the levels of tRNA modifications. The absence of TrmFO has a positive effect on the DNA synthesis under the nutrient-poor condition In vivo synthesis of CH2THF is catalyzed by SHMT using serine. Serine has to be synthesized by the cells when grown in the minimal synthetic medium. The methyl group provided by CH2THF is consumed not only for tRNA methylation by TrmFO but also for formation of dTMP by ThyX during DNA synthesis. Therefore, we considered that the consumption of CH2THF by TrmFO may have a negative effect on dTMP synthesis by ThyX. To test this, we analysed the extent of DNA synthesis in the wild-type and ΔtrmFO strains under the nutrient-poor condition. We added 3H-labelled serine to the cells when they were in early log phase (arrow in Fig. 2A), and measured the amount of 3H incorporation into the DNA 4 h after the addition of 3H-labelled serine. As shown in Fig. 3A, 3H-methyl groups derived from 3H-labelled serine had been more efficiently incorporated into the genomic DNA fraction from the ΔtrmFO strain than in the case of the wild-type. The 3H-radioisotope activities in the genomic DNA fractions were dramatically decreased by treatment with DNase I, but not with RNase A (Fig. 3A), confirming that no methylated RNA was contaminated in the samples. Is the slow-growth phenotype of the wild-type strain really attributed to non-optimal DNA synthesis? To address this, we added thymidine to cultures of wild-type cells in the early log phase (Fig. 3B), which resulted in accelerated growth (but the growth was slower than that of the ΔtrmFO strain). In contrast, the growth speed of ΔtrmFO strain in the presence of thymidine was almost the same as that in the absence of thymidine (data not shown). These results suggested that the ThyX-dependent formation of dTMP from dUMP is one of the important factors for the growth speed. Fig. 3 View largeDownload slide DNA synthesis under nutrient-poor conditions at 70°C. (A) At the point shown by an arrow in Fig. 3A, 3H-serine was added to the medium. After 4 h, cells were collected and 3H-methyl group incorporation into the DNA of the wild-type (black bars) and ΔtrmFO (white bars) strains were measured. The wild-type 3H-methyl group incorporation is set at 100%. The DNA fraction was treated with DNase I or RNase A, and the remaining 3H-activties in the acid-insoluble fractions were compared. P values are as follows: *, <0.005; **, <0.01; ***, not significant. (B) The growth of the wild-type strain at 70°C was accelerated by the addition of thymidine to the synthetic medium (n = 4). Fig. 3 View largeDownload slide DNA synthesis under nutrient-poor conditions at 70°C. (A) At the point shown by an arrow in Fig. 3A, 3H-serine was added to the medium. After 4 h, cells were collected and 3H-methyl group incorporation into the DNA of the wild-type (black bars) and ΔtrmFO (white bars) strains were measured. The wild-type 3H-methyl group incorporation is set at 100%. The DNA fraction was treated with DNase I or RNase A, and the remaining 3H-activties in the acid-insoluble fractions were compared. P values are as follows: *, <0.005; **, <0.01; ***, not significant. (B) The growth of the wild-type strain at 70°C was accelerated by the addition of thymidine to the synthetic medium (n = 4). Molecular ratio of SHMT, TrmFO and ThyX in vivo The consumption of CH2THF by ThyX and TrmFO on one hand and synthesis by SHMT on the other would be controlled by their relative abundance in the cell. To estimate the ratio of these proteins, we purified recombinant SHMT, TrmFO and ThyX (Fig. 4A) and prepared polyclonal antibodies against all three proteins. The polyclonal antibody fractions were used for estimation of their concentrations at early, mid, and late log phases during growth at 70°C: the cell extracts were prepared from the cells at early, mid, and late log phases, and the concentrations of their SHMT, TrmFO and ThyX were measured using their antibodies (Fig. 4B–E). The results are summarized in Fig. 4F. The amount of SHMT was slightly increased in mid-log phase, whereas that of ThyX was almost constant. Interestingly, the amount of TrmFO was increased during late log phase. Apart from these subtle differences, the ratio of SHMT, TrmFO and ThyX in the cells was almost unchanged (approximately 5:1:1.5) (Fig. 4F). Furthermore, we confirmed that the expression levels of SHMT and ThyX in the ΔtrmFO strain were the same as those in the wild-type strain (data not shown). Fig. 4 View largeDownload slide Subunit molecular ratio of SHMT, TrmFO and ThyX in living cells. (A) To prepare the polyclonal antibodies, SHMT, TrmFO and ThyX were purified and then analysed by 15% SDS- PAGE. The gel was stained with Coomassie Brilliant Blue. (B) The cell extracts were prepared from the wild-type strain cells at early-log (lane 1), mid-log (lanes 2 and 3), and late-log (lane 4) phases. The optical densities of cells at the points are shown at the bottom of panel F. 10 μg of proteins in each sample were analysed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (C) The concentrations of SHMT in the cell extracts were estimated by western blotting analysis. Lanes in the right side of the gel show the bands of standard (purified) SHMT to estimate the quantity. The band intensities were monitored by the fluorescence of secondary antibody (Alexa Fluor 488 anti-rabbit IgG). The amounts of TrmFO (D) and ThyX (E) were estimated by the same method. (F) The results of experiments are summarized. The relative abundances (subunit molecular ratios), which are indicated in the right side, are calculated from the band intensities: the abundance of TrmFO in the extract of mid-log phase cells (optical density at 600 nm is 0.6) is expressed as 1.00 (n = 4). Fig. 4 View largeDownload slide Subunit molecular ratio of SHMT, TrmFO and ThyX in living cells. (A) To prepare the polyclonal antibodies, SHMT, TrmFO and ThyX were purified and then analysed by 15% SDS- PAGE. The gel was stained with Coomassie Brilliant Blue. (B) The cell extracts were prepared from the wild-type strain cells at early-log (lane 1), mid-log (lanes 2 and 3), and late-log (lane 4) phases. The optical densities of cells at the points are shown at the bottom of panel F. 10 μg of proteins in each sample were analysed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (C) The concentrations of SHMT in the cell extracts were estimated by western blotting analysis. Lanes in the right side of the gel show the bands of standard (purified) SHMT to estimate the quantity. The band intensities were monitored by the fluorescence of secondary antibody (Alexa Fluor 488 anti-rabbit IgG). The amounts of TrmFO (D) and ThyX (E) were estimated by the same method. (F) The results of experiments are summarized. The relative abundances (subunit molecular ratios), which are indicated in the right side, are calculated from the band intensities: the abundance of TrmFO in the extract of mid-log phase cells (optical density at 600 nm is 0.6) is expressed as 1.00 (n = 4). In vitro competition between TrmFO and ThyX via CH2THF consumption We tested in vitro the relationship between TrmFO, ThyX and SHMT. For the measurement of ThyX activity, we adapted the TrmFO assay system (27) allowing analysis of 14C-incorporation into dTMP by thin-layer chromatography (Fig. 5A). In the absence of ThyX, 14C-CH2THF was synthesized by SHMT from 14C-labe1led serine: 14C-labelled serine was converted into 14C-labelled glycine by this reaction (Fig. 5A, left panel). Upon addition of ThyX, 14C-labelled dTMP was synthesized. To estimate the competitive effect of ThyX, we determined TrmFO activity under various concentrations of ThyX and SHMT while keeping the concentrations of TrmFO and tRNAPhe constant (Fig. 5B). Under mimicked natural conditions, with the ratio of SHMT, TrmFO and ThyX being 5:1:1.5, the labelling activity of TrmFO was about 40% (open square indicated by an arrow in Fig. 5B) of that in the absence of ThyX. The presence of ThyX interferes with, but does not completely abrogate TrmFO activity through the consumption of CH2THF (Fig. 5C). This result is in line with the observation that the m5U54 modification was near-fully introduced into the tRNA fraction even under the nutrient-poor condition (Fig. 2C). Because the concentrations of precursor tRNA and dUMP in living cells were unknown, we investigated the ThyX activities in the presence of various concentrations of dUMP and unmodified tRNA (isolated from the ΔtrmFO strain) (Fig. 5D). In the absence of tRNA, the ThyX activity was not dependent on the concentration of dUMP, showing that 7 μM dUMP in the mixture was sufficient for saturated dTMP synthesis (Fig. 5D). After increasing amounts of tRNA were added, the ThyX activity reduced because of competition for CH2THF by TrmFO (Fig. 5D). These in vitro experimental results confirmed that in vivo TrmFO and ThyX are dependent on the concentration of CH2THF, which, in a nutrient-poor environment, determines the speed of DNA-synthesis and thereby that of growth at 70°C. Fig. 5 View largeDownload slide TrmFO and ThyX interfere with each other through competition for CH2THF in vitro. (A) Thin layer chromatography assaying ThyX activity. In the absence of ThyX, SHMT synthesized 14C-labelled CH2THF from 14C-labelled serine (left panel). In the presence of ThyX, dUMP was methylated to 14C-labelled dTMP, which appeared to drive the conversion of 14C-labelled serine by SHMT (right panel). (B) The activity of TrmFO was reduced in the presence of various concentrations of ThyX and SHMT as measured by 3H-methyl group incorporation into unmodified tRNA (isolated from the ΔtrmFO strain) (n = 4). The arrow indicates ThyX activity when the subunit molecular ratio of SHMT, TrmFO and ThyX was 5:1:1.5, mimicking the in vivo situation. The concentration of TrmFO was 0.25 μM. (C) TrmFO activity at various concentrations of ThyX. Under the tested conditions, the presence of a surplus of ThyX did not out-compete TrmFO (0.25 μM) completely. In this experiment, 3H-labelled serine was used. (D) ThyX activity, measured as initial velocities of dTMP formation, under various concentrations of dUMP and tRNA (n = 4). The reaction mixture contained 1.25 μM SHMT, 0.25 μM TrmFO, 0.375 μM ThyX, 5 μM THF, 5 mM NADPH, 25 μM 14C-labelled serine, 0, 0.83, 7.5 or 25 μM tRNAPhe transcript, and 7, 35 or 170 μM dUMP. Fig. 5 View largeDownload slide TrmFO and ThyX interfere with each other through competition for CH2THF in vitro. (A) Thin layer chromatography assaying ThyX activity. In the absence of ThyX, SHMT synthesized 14C-labelled CH2THF from 14C-labelled serine (left panel). In the presence of ThyX, dUMP was methylated to 14C-labelled dTMP, which appeared to drive the conversion of 14C-labelled serine by SHMT (right panel). (B) The activity of TrmFO was reduced in the presence of various concentrations of ThyX and SHMT as measured by 3H-methyl group incorporation into unmodified tRNA (isolated from the ΔtrmFO strain) (n = 4). The arrow indicates ThyX activity when the subunit molecular ratio of SHMT, TrmFO and ThyX was 5:1:1.5, mimicking the in vivo situation. The concentration of TrmFO was 0.25 μM. (C) TrmFO activity at various concentrations of ThyX. Under the tested conditions, the presence of a surplus of ThyX did not out-compete TrmFO (0.25 μM) completely. In this experiment, 3H-labelled serine was used. (D) ThyX activity, measured as initial velocities of dTMP formation, under various concentrations of dUMP and tRNA (n = 4). The reaction mixture contained 1.25 μM SHMT, 0.25 μM TrmFO, 0.375 μM ThyX, 5 μM THF, 5 mM NADPH, 25 μM 14C-labelled serine, 0, 0.83, 7.5 or 25 μM tRNAPhe transcript, and 7, 35 or 170 μM dUMP. Methionine requirement under the nutrient-poor condition As shown in Fig. 2C, several tRNA modifications such as m1A, Cm, m7G, Gm, m1G and m2G were normally introduced into the tRNA fraction under the nutrient-poor condition. The limited nutrients were preferentially consumed in the tRNA modification systems. These methylated nucleosides are conferred by AdoMet-dependent tRNA methyltransferases (2). Thus, this observation suggested that AdoMet at least required for the tRNA methyltransferases was synthesized in the wild-type cells under the nutrient-poor condition. Considering the Km values of tRNA methyltransferases for AdoMet (52–54), at least more than 10 μM of AdoMet seemed to be required in the cells. Because methionine, a precursor of AdoMet, was not added to the minimal synthetic medium, methionine should be synthesized in the cells. Furthermore, methionine is required not only for AdoMet synthesis but also for many biological phenomena such as protein and phospholipid syntheses. In living cells, methionine is synthesized by methionine synthase (MetE) from homocysteine and N5-methyltetrahydoxylfolate (5-methylTHF), which is synthesized from CH2THF by MetF (55). Thus, considerable amount of CH2THF seemed to be consumed for the methionine synthesis under a nutrient-poor condition. To confirm this idea, we added methionine to the minimal synthetic medium (Supplementary Fig. S3). In the absence of methionine, the ΔtrmFO strain grew faster than the wild-type strain (Fig. 2A). When 100 or 300 μM of methionine was added to the medium, slight acceleration of growth of the ΔtrmFO strain was observed (data not shown). In contrast, when methionine was added to 1 mM, the wild-type and ΔtrmFO strains grew at almost the same speed (Supplementary Fig. S3). This result showed that the consumption of CH2THF in the methionine synthesis is a very important factor to decide the growth speed under the nutrient-poor condition. Because the addition of thymidine did not cause perfect recovery of growth speed (Fig. 3B), we concluded that the main reason of slow growth of the wild-type strain was the consumption of CH2THF for the methionine synthesis. Activities of TrmFO and MetF compete in in vitro experiments In T. thermophilus cells, methionine is synthesized from homocysteine and 5-methylTHF, which is converted from CH2THF by the reaction of MetF (56). Therefore, we tested whether the activity of MetF competes with the activity of TrmFO. It should be mentioned that T. thermophilus has the simplest MetF system (50, 56, 57): T. thermophilus MetF did not require other proteins for the activity and has only CH2THF-dependent NADH oxidase activity. T. thermophilus MetF was expressed in E. coli cells and purified (Fig. 6A). We confirmed the NADH oxidase activity. In brief, 0.4 μM SHMT, 5 μM THF, 25 μM serine and 200 μM NADH were incubated at 20°C for 10 min and then 3.0 μM MetF was added into the reaction mixture. As shown in Fig. 6B, NADH was consumed by the addition of MetF. This reaction was checked by formation of 14C-5-methylTHF from 14C-CH2THF using 14C-labelled serine (Fig. 6C). After these pilot experiments, TrmFO activities were measured at 60°C in the absence and presence of MetF (Fig. 6D). The activity of TrmFO was decreased in the presence of MetF. Thus, the activity of MetF competes with the activity of TrmFO in vitro. However, even in the presence of 10-fold larger amount of MetF, the methylation by TrmFO was still observed (bottom panels). This observation may explain the near-complete formation of m5U54 in tRNA under the nutrient-poor condition. In living cells, 5-methylTHF produced by MetF is converted to methionine by the reaction of MetE. Therefore, under the nutrient-poor condition, CH2THF may be consumed by the reactions of MetF and MetE more rapidly as compared with the results of in vitro experiments. Fig. 6 View largeDownload slide Activity of MetF competes with activity of TrmFO. (A) 8 μg of purified MetF was analysed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (B) NADH oxidase activity of MetF was monitored by decrease of absorbance at 340 nm. 0.4 μM SHMT, 5 μM THF, 25 μM serine and 200 μM NADH were incubated at 20°C for 10 min and then 3.0 μM MetF was added to the reaction mixture. (C) The formation of 5-methylTHF by MetF was checked using 14C-labelled serine. 14C-labelled CH2THF was synthesized by the activity of SHMT. The reaction was started by the addition of 14C-labelled serine. After 10 min, 3.0 μM MetF was added to the reaction mixture. (D) The activities of TrmFO in the absence and presence of MetF were analysed by 14C-methyl group incorporations into tRNAPhe transcript. The subunit molecular ratios of TrmFO and MetF are indicated at the left side. The gels were stained with methylene blue (left panels) and their autoradiograms were taken (right panels). Fig. 6 View largeDownload slide Activity of MetF competes with activity of TrmFO. (A) 8 μg of purified MetF was analysed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (B) NADH oxidase activity of MetF was monitored by decrease of absorbance at 340 nm. 0.4 μM SHMT, 5 μM THF, 25 μM serine and 200 μM NADH were incubated at 20°C for 10 min and then 3.0 μM MetF was added to the reaction mixture. (C) The formation of 5-methylTHF by MetF was checked using 14C-labelled serine. 14C-labelled CH2THF was synthesized by the activity of SHMT. The reaction was started by the addition of 14C-labelled serine. After 10 min, 3.0 μM MetF was added to the reaction mixture. (D) The activities of TrmFO in the absence and presence of MetF were analysed by 14C-methyl group incorporations into tRNAPhe transcript. The subunit molecular ratios of TrmFO and MetF are indicated at the left side. The gels were stained with methylene blue (left panels) and their autoradiograms were taken (right panels). Adenine requirement under the nutrient-poor condition CH2THF is used for purine synthesis in addition to tRNA methylation, dTMP synthesis and methionine synthesis. To investigate whether purine is an important factor for growth speed under the nutrient-poor condition, we added 100 μM of adenine into the synthetic medium. As shown in Supplementary Fig. S4, the growth speed of the wild-type strain was not changed. Thus, purine is not a rate-limiting factor of growth under the nutrient-poor condition. This result suggests that purine is supplied from the salvage system under the tested condition. Discussion The ΔtrmFO strain can grow faster than the wild-type strain in the synthetic medium at 70°C. As far as the authors know, until now, there is no report that a gene disruption of tRNA modification enzyme accelerates the growth. At the beginning of study, we assumed abnormal (or insufficient) modification(s) in tRNA. In the wild-type cells, TrmFO binds to precursor tRNA and then methylates U54. We supposed that this binding might have effect on the binding of other tRNA modification enzymes. However, contrary to our expectation, we could not find any abnormal modification in the tRNA fraction from the ΔtrmFO strain that was cultured under the nutrient-poor condition. The rapid growth of ΔtrmFO strain was observed only under the nutrient-poor condition: the wild-type and ΔtrmFO strains grow at the same speed in the nutrient-rich medium at 70°C (36). This observation prompted us to investigate the CH2THF consumption. The results of this study are summarized in Fig. 7. In the wild-type strain, CH2THF is mainly consumed by two metabolic pathways, dTMP and methionine syntheses. The presence of TrmFO decreases the speeds of dTMP and methionine syntheses. This is the reason of slow growth of the wild-type strain at 70°C under the nutrient-poor condition. Our in vitro experimental results revealed that TrmFO and ThyX, and TrmFO and MetF compete at several enzyme concentrations. The methionine synthesis pathway is complicatedly regulated: the expression levels of enzymes in the methionine metabolism are regulated by the concentrations of methionine and AdoMet (55). Therefore, in the case of methionine synthesis, our results of in vitro experiments may be not directly applied to in vivo phenomena. However it is clear that considerable amount of AdoMet is synthesized via methionine under the nutrient-poor condition because the majority of methylated nucleosides were normally introduced into the tRNA fraction. In contrast, in the case of ΔtrmFO strain, there is no consumption of CH2THF by TrmFO. Thus, the limited amount of CH2THF can be used preferentially for main pathways, and this advantage results in the rapid growth. Fig. 7 View largeDownload slide Consumption of CH2THF in T. thermophilus under the nutrient-poor condition at 70°C. Under the nutrient-poor condition, CH2THF is consumed by these pathways as described in the main text. In the case of ΔtrmFO strain, there is no consumption of CH2THF by TrmFO. Fig. 7 View largeDownload slide Consumption of CH2THF in T. thermophilus under the nutrient-poor condition at 70°C. Under the nutrient-poor condition, CH2THF is consumed by these pathways as described in the main text. In the case of ΔtrmFO strain, there is no consumption of CH2THF by TrmFO. T. thermophilus lives in hot springs, in which nutrients such as amino acids are limited like in the nutrient-poor condition of our tests. Our experimental results revealed that the limited nutrients in the nutrient-poor condition are preferentially consumed in the tRNA modification systems. Modified nucleosides in tRNA are required for accurate and efficient protein synthesis (2, 25). If the tRNA modifications are insufficient, it directly causes the defects in the protein synthesis. For example, the absence of m1G37 modification in tRNA causes the frame-shift error (58) and incorrect recognition by aminoacyl-tRNA synthetase (59). Furthermore, mnm5U derivatives at position 34 are required for correct reading of codons and maintenance of reading frame (60–64). The errors in protein synthesis abolish numerous ATP, GTP and amino acids. Therefore, the modifications in tRNA may be preferentially introduced. The m5U54 modification by TrmFO is not essential for viability of T. thermophilus and is required only for the assistant of Ψ55 modification at low temperatures. T. thermophilus would be exposed to a wide range of temperatures, for example when hot spring water is cooled down by influx of river water or snow fall, while the hot spring can flow over or experience an eruption of hot water. The fine tuning of protein synthesis through the network of modified nucleosides in tRNA and tRNA modification enzymes seems to be very important for the survival of T. thermophilus. The physiological reason why TrmFO uses CH2THF is unclear. The m5U54 itself can be synthesized from AdoMet. Indeed, several eubacteria including E. coli possess AdoMet-dependent tRNA (m5U54) methyltransferase (TrmA) (13). Because AdoMet is synthesized by multiple steps from CH2THF under the nutrient-poor condition, consumption of AdoMet seems to be in the same situation as that of CH2THF from the viewpoint of one carbon metabolism. AdoMet is consumed in many metabolic pathways such as lipid synthesis, DNA methylation and polyamine synthesis in addition to the RNA modification. T. thermophilus produces various polyamines including long and branched polyamines (65). Two proteins, triamine/agmatine aminopropyltransferase (SpeE) and S-adenosyl-l-methionine decarboxylase-like protein 1 (SpeD1), are required for biosynthesis of long and branched polyamines from spermidine (66, 67). The gene-deletion strains of speE and speD1 could not grow in the synthetic medium at above 75°C (67). In contrast, the wild-type strain can grow at 80°C under the nutrient-poor condition. These observations suggest that considerable amount of AdoMet is consumed in the biosynthetic pathway of polyamines under the nutrient-poor condition. The rapid growth of ΔtrmFO strain under the nutrient-poor condition was only observed at 70°C. At 50°C, the wild-type and ΔtrmFO strains grew at nearly the same speed (data not shown). These observations suggest that the rate-limiting factor(s) for growth speed may be changed according to the culture temperatures. At the optimal temperature (70°C), the speed of dTMP and methionine syntheses seem to have the direct effect on the growth speed. Finally, this study revealed that tRNA modification systems in T. thermophilus preferentially consume the limited nutrients (CH2THF and AdoMet) under the nutrient-poor condition. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements We thank previous collaborators (Dr Chie Tomikawa in Ehime University, Japan and Prof Satoko Yoshizawa in CNRS, France) for valuable discussion. Funding This work was supported by a Grant-in-Aid for JSPS Fellows 26-8015(to R.Y.), a Grant-in-Aid for Scientific Researches 23350081 and 16H04763 (to H.H.) and a Grant-in-Aid for Exploratory Research 24655156 (to H.H.) from the Japan Society for the Promotion of Science (JSPS). Conflict of Interest None declared. 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( 2017) Long and branched polyamines are required for maintenance of the ribosome, tRNAHis and tRNATyr in Thermus thermophilus cells at high temperatures. Genes Cells  22, 628– 645 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations AdoMet S-adenosyl-l-methionine CH2THF N5, N10-methylenetetrahydrofolate cmnm5U 5-carboxymethylaminomethyluridine D dihydrouridine Gm 2′-O-methylguanosine m1A 1-methyladenosine 5-methylTHF N5-methyltetrahydroxylfolate m7G 7-methylguanosine MnmEG MnmE and MnmG complex mnm5U 5-methylaminomethyluridine m5s2U 5-methyl-2-thiouridine m5U 5-methyluridine PAGE polyacrylamide gel electrophoresis Ψ pseudouridine THF tetrahydrofolate © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Consumption of N5, N10-methylenetetrahydrofolate in Thermus thermophilus under nutrient-poor condition

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
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10.1093/jb/mvy037
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29538705
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Abstract

Abstract TrmFO catalyzes the formation of 5-methyluridine at position 54 in tRNA and uses N5, N10-methylenetetrahydrofolate (CH2THF) as the methyl group donor. We found that the trmFO gene-disruptant strain of Thermus thermophilus, an extremely thermophilic eubacterium, can grow faster than the wild-type strain in the synthetic medium at 70°C (optimal growth temperature). Nucleoside analysis revealed that the majority of modifications were appropriately introduced into tRNA, showing that the limited nutrients are preferentially consumed in the tRNA modification systems. CH2THF is consumed not only for tRNA methylation by TrmFO but also for dTMP synthesis by ThyX and methionine synthesis by multiple steps including MetF reaction. In vivo experiment revealed that methylene group derived from serine was rapidly incorporated into DNA in the absence of TrmFO. Furthermore, the addition of thymidine to the medium accelerated growth speed of the wild-type strain. Moreover, in vitro experiments showed that TrmFO interfered with ThyX through consumption of CH2THF. Addition of methionine to the medium accelerated growth speed of wild-type strain and the activity of TrmFO was disturbed by MetF. Thus, the consumption of CH2THF by TrmFO has a negative effect on dTMP and methionine syntheses and results in the slow growth under a nutrient-poor condition. DNA synthesis, MetF, RNA modification, ThyX, TrmFO To date more than 90 modified nucleosides have been found in tRNA (1) and biosynthesis pathways of these modified nucleosides often include a methylation step(s) by a tRNA methyltransferase(s) (2). In general, tRNA methyltransferases consume S-adenosyl-l-methionine (AdoMet) as the methyl group donor. However, exceptionally, two eubacterial tRNA modification enzymes, TrmFO (3) and the MnmE (4, 5) and MnmG complex (MnmEG) (6, 7), use N5, N10-methylenetetrahydrofolate (CH2THF) as the methyl group donor (Supplementary Fig. S1A). The trmFO genes are present in the genomes of almost all gram-positive and 70% of gram-negative bacteria (3) whereas the mnmE and mnmG genes have been found widely in eubacterial genomes (8). Although both TrmFO and MnmEG are CH2THF- and FAD-dependent tRNA modification enzymes, these enzymes modify uridines at different positions in tRNA: TrmFO is required for the formation of 5-methyluridine at position 54 (m5U54) in the T-loop, whereas MnmEG is needed for the 5-methylaminomethyluridine (mnm5U34) and the 5-carboxymethylaminomethyluridine (cmnm5U34) modifications at position 34 in the anticodon loop (Supplementary Fig. S1B). The m5U54 has been commonly found in tRNAs from eukaryotes, eubacteria and some archaea (2, 9–11) while mnm5U34 and cmnm5U34 derivatives have been found in limited numbers of eubacterial tRNA species: in the case of Escherichia coli, only six tRNA species possess mnm5U34 or cmnm5U34 derivatives (12). Therefore, m5U54 formation is the main pathway among tRNA modification systems in which CH2THF is consumed (Supplementary Fig. S1). It should be mentioned that some gram-negative bacteria including E. coli have an AdoMet-dependent tRNA (m5U54) methyltransferase (TrmA) (13, 14). Therefore, in the case of E. coli, for tRNA methylation, CH2THF is only used by MnmEG. Furthermore, Mycoplasma capricolum exceptionally possesses a folate- and flavin-dependent rRNA methyltransferase (15): this methyltransferase has not been found in the other eubacteria. The m5U54 forms a reverse Hoogsteen base pair with A58 in tRNA (16–18): A58 is often modified to 1-methyladenosine at position 58 (m1A58) (19). The m5U54-A58 (or m1A58) base pair stacks with the G53-C61 base pair in the T-arm and as a result, the L-shaped tRNA structure is stabilized. Therefore, m5U54 is widely found in tRNAs in three domains of life (1, 2, 9). In thermophilic eubacteria such as Thermus thermophilus (20–22) and Aquifex aeolicus (23), and some thermophilic archaea (24), m5U54 is further modified to 5-methyl-2-thiouridine (m5s2U54). The 2-thiomodification at position 54 shifts the equilibrium of ribose puckering to the C3′-endo form and the introduced sulphur atom enhances the stacking effect between the m5U54-A58 (or m1A58) and G53-C61 base pairs (25). In 2009, we determined the X-ray crystal structure of T. thermophilus TrmFO (26). In 2012, we devised an in vitro system to assay TrmFO activity and elucidated the substrate tRNA recognition mechanism of TrmFO: TrmFO recognizes the conserved U54U55C56 sequence and G53-C61 base pair in the T-arm structure (27). The hypothetical catalytic mechanism of the reaction by TrmFO has been proposed by Hamdane et al. (28–30). Thermus thermophilus can grow at wide ranges of temperature (50–83°C) (31). In recent studies (32–36), we reported that modified nucleotides in tRNA and tRNA modification enzymes in T. thermophilus form a network and that this network changes the degrees of modification in tRNAs according to the temperature of the culture. The protein synthesis system in T. thermophilus can adapt to the changes in temperature through alteration of structural flexibility (rigidity) of tRNA (22, 25, 32–34, 36). Figure 1A shows the modifications and responsible enzymes in T. thermophilus tRNAPhe (32, 37). The network at low temperatures (below 55°C) is illustrated in Fig. 1B. For example, at 50°C, the pseudouridine at position 55 (Ψ55) in tRNA prevents excess 2′-O-methylguanosine at position 18 (Gm18) modification by TrmH and m1A58 modification by TrmI (33). When tRNA (Ψ55) synthase gene (truB) was disrupted, the modification level of m5s2U54 in tRNA at 50°C increased (33). Because the m1A58 modification is required for sulphur transfer in the formation of m5s2U54 at 80°C (22), the negative effect of Ψ55 on the m5s2U54 formation at 50°C may be indirectly caused by the effect of m1A58. Totally, the presence of Ψ55 prevents excessive formation of Gm18, m1A58 and m5s2U54 at 50°C (33). Therefore, the Ψ55 modification contributes to maintaining the flexibility of tRNA at low temperatures. In contrast, at 80°C, the modification of G46 by TrmB (to m7G46 (32, 38–40)) accelerates formation of tRNA modifications, Gm18 (by TrmH (41–43)), m1A58 (by TrmI (19, 34)) and m1G37 (by TrmD (44)), which prevent structural disruption of tRNA at high temperatures (32). At low temperatures (below 55°C), the effect of m7G46 on other modifications is unclear (Fig. 1B). Furthermore, we reported that the dihydrouridine at position 20 (D20) formed by Dus did not have an effect on the network (35). Moreover, we investigated the role of m5U54 modification in the network: the absence of m5U54 modification in tRNA causes the increase of Gm18 and slight decrease of m1A58 at 50°C (36). Thus, the m5U54 modification in tRNA coordinately works with the Ψ55 modification to maintain the balance of tRNA modifications at low temperatures (Fig. 1B). The trmFO gene disruptant (ΔtrmFO) strain showed slight growth retardation in the nutrient-rich medium at 50°C (36). Fig. 1 View largeDownload slide Transfer RNA modification network in T. thermophilus at low temperatures. (A) The modified nucleosides and responsible enzymes in T. thermophilus tRNAPhe are illustrated. Bold letters show the modified nucleosides and positions. The responsible enzymes are connected by arrows. Abbreviations of modified nucleosides are as follows; m2G, N2-methylguanosine; s4U, 4-thiouridine; Gm, 2′-O-methylguanosine; D, dihydrouridine; i6A, N6-isopetenyladenosine; ms2i6A, 2-methylthio-N6-isopentenyladenosine; Ψ, pseudouridine; m7G, 7-methylguanosine; m5U, 5-methyluridine; m5s2U, 5-methyl-2-thiouridine; m1A, 1-methyladenosine. (B) The network between the modified nucleosides in tRNA and tRNA modification enzymes in T. thermophilus at low temperatures is illustrated. As described in the main text, this network contributes to maintaining the balance of modification levels in tRNA. Fig. 1 View largeDownload slide Transfer RNA modification network in T. thermophilus at low temperatures. (A) The modified nucleosides and responsible enzymes in T. thermophilus tRNAPhe are illustrated. Bold letters show the modified nucleosides and positions. The responsible enzymes are connected by arrows. Abbreviations of modified nucleosides are as follows; m2G, N2-methylguanosine; s4U, 4-thiouridine; Gm, 2′-O-methylguanosine; D, dihydrouridine; i6A, N6-isopetenyladenosine; ms2i6A, 2-methylthio-N6-isopentenyladenosine; Ψ, pseudouridine; m7G, 7-methylguanosine; m5U, 5-methyluridine; m5s2U, 5-methyl-2-thiouridine; m1A, 1-methyladenosine. (B) The network between the modified nucleosides in tRNA and tRNA modification enzymes in T. thermophilus at low temperatures is illustrated. As described in the main text, this network contributes to maintaining the balance of modification levels in tRNA. During the course of study (36), we found that the ΔtrmFO strain can grow faster than the wild-type strain in the synthetic medium even at 70°C (optimal growth temperature for T. thermophilus) (Fig. 2A). CH2THF is consumed by other metabolic pathways such as dTMP, purine and methionine syntheses (see Fig. 7). CH2THF is the methyl group donor of thymidylate synthase (ThyX), which catalyzes the methylation of dUMP to form dTMP (45, 46). Because CH2THF is labile and the amount of CH2THF supplied by serine hydroxymethyltransferase (SHMT) in living cells is limited, the synthesis of dTMP by ThyX is expected to be the rate-limiting step in DNA synthesis (47). We assumed that the consumption of CH2THF by TrmFO might have an effect on the DNA synthesis and/or other metabolisms of one-carbon via folate derivatives in living cells. In this paper, we report that the consumption of CH2THF by TrmFO under a nutrient-poor condition have a negative effect on the DNA and methionine syntheses. Fig. 2 View largeDownload slide The trmFO gene-disruptant (ΔtrmFO) strain can grow faster than the wild-type strain under the nutrient-poor condition at 70°C. (A) The growth of ΔtrmFO strain (open circles) was faster than that of the wild-type strain (filled circles) in the synthetic minimal medium at 70°C (the data are averages of six independent experiments. (n = 6)). The arrow shows the time point that 3H-serine was added to the medium in the experiment in Fig. 4A. (B) Modified nucleosides in the tRNA fraction from the wild-type strain cells, which were cultured in the nutrient-rich medium at 70°C. Unknown compound (*) from phosphodiesterase, which was used for the digestion of tRNAs, overlapped with the peak of m5U. Furthermore, s2U overlapped with the peak of G. Details were described in our previous paper (36). (C) Modified nucleosides in the tRNA fraction from the wild-type strain cells, which were cultured in the nutrient-poor medium at 70°C. (D) The tRNA fractions from the wild-type (left) and ΔtrmFO (right) strain cells, which were cultured under the nutrient-poor condition, were methylated by E. coli TrmA to estimate the m5U modification levels in tRNA. Fig. 2 View largeDownload slide The trmFO gene-disruptant (ΔtrmFO) strain can grow faster than the wild-type strain under the nutrient-poor condition at 70°C. (A) The growth of ΔtrmFO strain (open circles) was faster than that of the wild-type strain (filled circles) in the synthetic minimal medium at 70°C (the data are averages of six independent experiments. (n = 6)). The arrow shows the time point that 3H-serine was added to the medium in the experiment in Fig. 4A. (B) Modified nucleosides in the tRNA fraction from the wild-type strain cells, which were cultured in the nutrient-rich medium at 70°C. Unknown compound (*) from phosphodiesterase, which was used for the digestion of tRNAs, overlapped with the peak of m5U. Furthermore, s2U overlapped with the peak of G. Details were described in our previous paper (36). (C) Modified nucleosides in the tRNA fraction from the wild-type strain cells, which were cultured in the nutrient-poor medium at 70°C. (D) The tRNA fractions from the wild-type (left) and ΔtrmFO (right) strain cells, which were cultured under the nutrient-poor condition, were methylated by E. coli TrmA to estimate the m5U modification levels in tRNA. Materials and Methods Materials [Methyl-14C]-AdoMet (1.95 GBq/mmol), [methyl-3 H]-AdoMet (2.47 TBq/mmol), l-[3 H(G)]-serine (1.48 TBq/mmol), l-[14C(U)]-serine (1.85 GBq/mmol) and [methyl-3 H]-thymidine (3.33 TBq/mmol) were purchased from Perkin Elmer Life Science. Standard 5-methyluridine (m5U), tetrahydrofolate (THF), adenine and N5-methyltetrahydoxylfolate (5-methylTHF) were obtained from Sigma Aldrich. DE81 filter (code number 3658-323) and 3 MM filter were purchased from Whatman. DNA oligonucleotides were purchased from Invitrogen. HiTrap Heparin HP, HiTrap Blue HP, Hi Trap Q HP and Q-Sepharose columns were purchased from GE Healthcare. The other chemical reagents were of analytical grade. Strain and media The culture source of T. thermophilus HB8 and the procedure for culturing this strain on synthetic minimal medium were a kind gift from Dr Tairo Oshima (Kyowa-kako, Japan). Nutrient-rich medium contained 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, pH 7.5 (adjusted by NaOH) and the medium was supplemented with 0.35 mM CaCl2 and 0.17 mM MgCl2 after autoclaving. The synthetic medium contained the following components (per l litter): 20 g sucrose, 20 g sodium glutamate, 0.5 g K2HPO4, 0.25 g KH2PO4, 2 g NaCl, 0.5 g (NH4)2SO4, 0.1 mg biotin, 1 mg thiamine, 125 mg MgCl2-6H2O, 25 mg CaCl2-2H2O, 1.15 mg NaMoO4-2H2O, 0.1 mg VOSO4-xH2O, 0.5 mg MnCl2-4H2O, 0.06 mg ZnSO4-7H2O, 0.01 mg CuSO4-5H2O, 0.8 mg CoCl2-6H2O, 0.02 mg NiCl2-6H2O, 0.03 µl H2SO4 and 6 mg FeSO4-7H2O. To make plates, gellan gum (Wako Pure Chemicals) was added to the medium (final concentration, 1.5%). Preparation of tRNA mixtures and nucleoside analysis Total RNA was prepared as described in our recent report (48). The tRNA fraction was further purified by 10% polyacrylamide gel containing 7 M urea electrophoresis [PAGE (7 M urea)]. Nucleoside analysis was performed after complete digestion of tRNA with phosphodiesterase, RNase A and bacterial alkaline phosphatase as described previously (32). In vitro methylation of tRNA Transfer RNA mixture (0.20 A260units) from the wild-type or ΔtrmFO strain was methylated by 5 μg of TrmA and 30 μM of 14C-AdoMet at 37°C for 3 h in 50 μl buffer A [50 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 6 mM 2-mercaptoethanol and 50 mM KCl]. The expression and purification of recombinant E. coli TrmA were previously reported (33). The incorporation of the 14C-methyl group was monitored with a liquid scintillation counter (LSC) using a filter assay. Incorporation of radio-labelled serine A total of 370 kBq of 3H-labelled serine was added to 10 ml of culture which was further incubated at 70°C for 4 h. The cells were collected by centrifugation at 6100 × g at 4°C for 2 min, washed with 500 μl of fresh medium and collected. The genomic DNA fraction was extracted from these cells by means of the NucleoSpin Tissue kit (Takara) according to the manufacturer’s instructions and 1 μg of genomic DNA was spotted onto a 3MM filter, and 3H-methyl group incorporation was measured with a LSC. RNase A and DNase I treatments were performed as follows: 400 ng of the genomic DNA was treated with 40 μg of RNase A (Nacalai Tesque) or 20 units of DNase I (Roche) in 30 μl of buffer A at 37°C for 60 min, and the 3H-methyl group content in the sample was measured with a LSC. Expression and purification of recombinant proteins for TrmFO activity measurement The E. coli expression systems and purification procedures used for TrmFO and SHMT were as described previously (26). Expression and purification of ThyX Thermus thermophilus ThyX (TTHA1096)-pET11b expression vector was purchased from RIKEN Biological Resource Center (49) and ThyX was expressed using E. coli BL21 (DE3) Rosetta2 (Novagen) according to the manufacture’s manual. In short: harvested cells (2.8 g) were suspended in 15 ml of buffer A, in which 1 tablet of protease inhibitor cocktail (Roche) was dissolved, and lysed in an ultrasonic disruptor (model VCX-500, Sonics and Materials., Inc., USA) at 4°C. The cell debris was removed by centrifugation at 6000 × g at 4°C for 20 min. The supernatant was heated at 70°C for 20 min, and denatured proteins were removed by centrifugation at 9000 × g at 4°C for 20 min. The supernatant was loaded onto a Hi Trap Q HP column (5 ml) equilibrated with buffer A. ThyX was eluted by a linear gradient of 50 mM–1 M KCl in buffer A. The eluted ThyX fractions were collected, dialyzed against buffer A using a Spectra/Por 7 dialysis membrane (MWCO: 10,000, code 132119, Spectrum Laboratories) at 4°C, and then loaded onto a Hi Trap Blue HP column (5 ml). The bound proteins were eluted by a linear gradient of 50 mM–1.5 M KCl in buffer A. The ThyX fractions were collected and concentrated in a Vivaspin 20-10K ultrafiltration membrane device (GE Healthcare). The concentrated ThyX fraction (3 ml) was loaded onto a HiLoad Superdex 75 preparation-grade gel-filtration column (120 ml, GE Healthcare) equilibrated with buffer A containing 400 mM KCl. The eluted ThyX fractions were collected, dialyzed against buffer A, and concentrated. The purified ThyX (5.7 mg) was mixed with an equivalent volume of 100% glycerol and stocked at −30°C. The purity was checked by 15% SDS-PAGE. Preparation of polyclonal antibodies and western blotting analysis Customized rabbit anti-TrmFO, anti-SHMT and anti-ThyX serums were prepared by Kitayama Labes, Japan, using the purified recombinant proteins as the antigens. Polyclonal antibody fractions were prepared using an Econo-pac serum IgG purification kit (Bio-Rad). Western blotting analysis was performed as described in our recent report (35). Measurement of ThyX activity To visualize the methyl group transfer reaction, we set up a TLC assay system for ThyX. The reaction mixtures (0.4 μM SHMT, 5 μM THF, 5 mM NADPH, 25 μM 14C-labelled serine and 170 μM dUMP in buffer A) were incubated at 60°C and samples were taken at the indicated time points. The methyl transfer reaction was started with the addition of ThyX (final concentration, 0.5 μM). The reaction was stopped by the addition of an equal volume of phenol/chloroform (1:1), and the aqueous phases were recovered. Aliquots (1.5 μl) were spotted onto a thin-layer plate (TLC Cellulose F, code 1.05565.0001, Merck) and the sample was separated by a solvent system of isopropyl alcohol: HCl:H2O, 70:15:15, v/v/v. The 14C-labelled compounds were visualized by Typhoon FLA 7000. The mobility of standard markers was detected by irradiation of UV at 254 nm and by the ninhydrin reaction. Disturbance competition assay between TrmFO and ThyX The reaction mixture containing various concentration of SHMT, 5 μM THF, 5 mM NADPH, 25 μM 3H-labelled serine, 5.6 μM tRNAPhe transcript, and 170 μM dUMP in buffer A was pre-incubated at 60°C for 10 min. The methyl transfer reaction was started by the addition of TrmFO and ThyX, and the reaction mixture was incubated at 60°C for 10 min. Aliquots of 25 μl were spotted onto a DE81 paper. The filter was washed with 250 mM Na2HPO4 for at least five times at room temperature. The methyl group incorporation into tRNAPhe transcript was measured with a LSC. Cultures in synthetic medium with methionine or adenine Methionine was dissolved in water (final concentration, 50 mg/ml), and then pH was adjusted by 5 M NaOH to 7.0. The dissolved methionine was directly added to the synthetic medium. The culture was performed at 70°C. Adenine was directly added to the synthetic medium (final concentration, 100 μM). Expression and purification of MetF Thermus thermophilus MetF (TTHA0327)-pET11b expression vector was purchased from RIKEN Biological Resource Center (49) and MetF was expressed using E. coli Rosetta2 (DE3). Purification of MetF was performed according to the reference (50) with slight modifications. In brief, cells (2.0 g) was suspended in 10 ml of buffer B (50 mM potassium phosphate (pH 7.2), 200 mM NaCl), in which 1 tablet of protease inhibitor cocktail (Roche) was dissolved. The cells were disrupted in an ultrasonic disruptor (model VCX-500, Sonics and Materials, Inc., USA) at 4°C. The cell debris was removed by centrifugation at 20,000 × g at 4°C for 15 min. The supernatant was heated at 70°C for 20 min, and denatured proteins were removed by centrifugation at 8000 x g at 4°C for 20 min. The supernatant was loaded onto a Q-Sepharose column (volume, 5 ml) and then separated by a NaCl linear gradient (200–1000 mM). The eluted sample was dialyzed against buffer A. After dialysis, the precipitant was removed by centrifugation at 8000 × g at 4°C for 20 min. The supernatant fraction (33.2 mg of purified MetF) was concentrated in a Vivaspin 20-10K ultrafiltration membrane device. Activity measurement of MetF NADH oxidase activity of MetF was measured by monitoring of decrease of absorbance at 340 nm at 20°C as follows. 0.4 μM SHMT, 5 μM THF, 200 μM NADH and 25 μM serine in 1 ml of buffer A were pre-incubated at 20°C for 10 min, and then MetF was added (final concentration, 3 μM). Formation of 14C-labelled 5-methylTHF was confirmed by thin-layer chromatography using 14C-labelled serine: To separate CH2THF and 5-methylTHF, the solvent system (phenol-water: conc. ammonia-water, 99) was used. The mobility of 5-methylTHF was checked by UV at 360 nm irradiation using the standard compound. Competitive assay between TrmFO and MetF A total of 0.4 μM SHMT, 5 μM THF, 5 mM NADH, 5 mM NADPH, 1 mM FAD, 25 μM tRNAPhe transcript, 0.75 μM TrmFO, 25 μM 14C-labelled serine, and 0, 0.75, 2.25 or 7.5 μM MetF in 200 μl of buffer A were incubated at 60°C and then 35 μl of aliquot was taken at 0, 10, 20, 30 and 40 min period. Transfer RNA transcripts were recovered by phenol-chloroform treatment and ethanol precipitation, and then analysed by 10% PAGE (7 M urea). The gels were stained with methylene blue and 14C-methyl group incorporations were monitored by Typhoon FLA 7000. Results The ΔtrmFO strain grows faster than the wild-type under nutrient-poor condition at 70°C Under a nutrient-poor condition (minimal synthetic medium), the ΔtrmFO strain grew faster than the wild-type strain at 70°C (Fig. 2A). Because the m5U54 modification in tRNA supports the maintenance of balance of tRNA modifications only at low temperatures (36), this phenomenon at 70°C was not simply explainable by the effect of m5U54 in the network. At the start of this study, we assumed that some modifications are abnormally (or insufficiently) introduced into tRNA under the nutrient-poor condition. To verify this idea, we purified tRNA fractions from the wild-type, which were cultured under nutrient-rich and -poor conditions at 70°C. The modified nucleosides in these tRNA fractions were compared by HPLC C18-reverse-phase column chromatography (Fig. 2B and C). Contrary to our expectation, we could not find any abnormal modifications in the tRNA fraction from the cells cultured under the nutrient-poor condition (Fig. 2B and C): the majority of tRNA modifications (Ψ, m1A, Cm, m7G, m5U, s4U, Gm, m1G, m2G and m5s2U) were sufficiently introduced into the tRNA fraction, showing that the limited nutrients are preferentially consumed in the tRNA modification systems. It should be mentioned that mnm5U derivatives were not detected in Fig. 2B and C because they are hydrophobic and are contained in the limited tRNA species. Furthermore, we checked the modification levels of tRNAs from the ΔtrmFO strain that were cultured in the synthetic minimal medium at 70°C (Supplementary Fig. S2). As shown in Supplementary Fig. S2, we could not find any abnormal modification in this fraction. The peak of m5U overlapped with the peak of unknown compound (asterisks in Fig. 2 and Supplementary Fig. S2) derived from phosphodiesterase, which was used for digestion of tRNA. Furthermore, the s2U and m5s2U peaks overlapped with the peaks of G and m2G, respectively. Therefore, to know the m5U54 and m5s2U54 modification levels, we measured the methylation capacity in the tRNA fractions of wild-type and ΔtrmFO strains by TrmA [E. coli tRNA (m5U54) methyltransferase (13, 14)] and 14C-labelled AdoMet. E. coli TrmA does not catalyze the exchange reaction of methyl group in tRNA (51) and is able to convert s2U54 to m5s2U54 (36). The tRNA fraction from the ΔtrmFO strain did not contain the m5U54 and m5s2U54 modifications (positive control). As shown in Fig. 2D, the 5-carbon in U54 in the tRNA fraction from the wild-type strain cultured under the nutrient-poor condition was near-fully methylated. These experimental results suggested that the rapid growth of ΔtrmFO strain under the nutrient-poor condition was not explainable by the levels of tRNA modifications. The absence of TrmFO has a positive effect on the DNA synthesis under the nutrient-poor condition In vivo synthesis of CH2THF is catalyzed by SHMT using serine. Serine has to be synthesized by the cells when grown in the minimal synthetic medium. The methyl group provided by CH2THF is consumed not only for tRNA methylation by TrmFO but also for formation of dTMP by ThyX during DNA synthesis. Therefore, we considered that the consumption of CH2THF by TrmFO may have a negative effect on dTMP synthesis by ThyX. To test this, we analysed the extent of DNA synthesis in the wild-type and ΔtrmFO strains under the nutrient-poor condition. We added 3H-labelled serine to the cells when they were in early log phase (arrow in Fig. 2A), and measured the amount of 3H incorporation into the DNA 4 h after the addition of 3H-labelled serine. As shown in Fig. 3A, 3H-methyl groups derived from 3H-labelled serine had been more efficiently incorporated into the genomic DNA fraction from the ΔtrmFO strain than in the case of the wild-type. The 3H-radioisotope activities in the genomic DNA fractions were dramatically decreased by treatment with DNase I, but not with RNase A (Fig. 3A), confirming that no methylated RNA was contaminated in the samples. Is the slow-growth phenotype of the wild-type strain really attributed to non-optimal DNA synthesis? To address this, we added thymidine to cultures of wild-type cells in the early log phase (Fig. 3B), which resulted in accelerated growth (but the growth was slower than that of the ΔtrmFO strain). In contrast, the growth speed of ΔtrmFO strain in the presence of thymidine was almost the same as that in the absence of thymidine (data not shown). These results suggested that the ThyX-dependent formation of dTMP from dUMP is one of the important factors for the growth speed. Fig. 3 View largeDownload slide DNA synthesis under nutrient-poor conditions at 70°C. (A) At the point shown by an arrow in Fig. 3A, 3H-serine was added to the medium. After 4 h, cells were collected and 3H-methyl group incorporation into the DNA of the wild-type (black bars) and ΔtrmFO (white bars) strains were measured. The wild-type 3H-methyl group incorporation is set at 100%. The DNA fraction was treated with DNase I or RNase A, and the remaining 3H-activties in the acid-insoluble fractions were compared. P values are as follows: *, <0.005; **, <0.01; ***, not significant. (B) The growth of the wild-type strain at 70°C was accelerated by the addition of thymidine to the synthetic medium (n = 4). Fig. 3 View largeDownload slide DNA synthesis under nutrient-poor conditions at 70°C. (A) At the point shown by an arrow in Fig. 3A, 3H-serine was added to the medium. After 4 h, cells were collected and 3H-methyl group incorporation into the DNA of the wild-type (black bars) and ΔtrmFO (white bars) strains were measured. The wild-type 3H-methyl group incorporation is set at 100%. The DNA fraction was treated with DNase I or RNase A, and the remaining 3H-activties in the acid-insoluble fractions were compared. P values are as follows: *, <0.005; **, <0.01; ***, not significant. (B) The growth of the wild-type strain at 70°C was accelerated by the addition of thymidine to the synthetic medium (n = 4). Molecular ratio of SHMT, TrmFO and ThyX in vivo The consumption of CH2THF by ThyX and TrmFO on one hand and synthesis by SHMT on the other would be controlled by their relative abundance in the cell. To estimate the ratio of these proteins, we purified recombinant SHMT, TrmFO and ThyX (Fig. 4A) and prepared polyclonal antibodies against all three proteins. The polyclonal antibody fractions were used for estimation of their concentrations at early, mid, and late log phases during growth at 70°C: the cell extracts were prepared from the cells at early, mid, and late log phases, and the concentrations of their SHMT, TrmFO and ThyX were measured using their antibodies (Fig. 4B–E). The results are summarized in Fig. 4F. The amount of SHMT was slightly increased in mid-log phase, whereas that of ThyX was almost constant. Interestingly, the amount of TrmFO was increased during late log phase. Apart from these subtle differences, the ratio of SHMT, TrmFO and ThyX in the cells was almost unchanged (approximately 5:1:1.5) (Fig. 4F). Furthermore, we confirmed that the expression levels of SHMT and ThyX in the ΔtrmFO strain were the same as those in the wild-type strain (data not shown). Fig. 4 View largeDownload slide Subunit molecular ratio of SHMT, TrmFO and ThyX in living cells. (A) To prepare the polyclonal antibodies, SHMT, TrmFO and ThyX were purified and then analysed by 15% SDS- PAGE. The gel was stained with Coomassie Brilliant Blue. (B) The cell extracts were prepared from the wild-type strain cells at early-log (lane 1), mid-log (lanes 2 and 3), and late-log (lane 4) phases. The optical densities of cells at the points are shown at the bottom of panel F. 10 μg of proteins in each sample were analysed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (C) The concentrations of SHMT in the cell extracts were estimated by western blotting analysis. Lanes in the right side of the gel show the bands of standard (purified) SHMT to estimate the quantity. The band intensities were monitored by the fluorescence of secondary antibody (Alexa Fluor 488 anti-rabbit IgG). The amounts of TrmFO (D) and ThyX (E) were estimated by the same method. (F) The results of experiments are summarized. The relative abundances (subunit molecular ratios), which are indicated in the right side, are calculated from the band intensities: the abundance of TrmFO in the extract of mid-log phase cells (optical density at 600 nm is 0.6) is expressed as 1.00 (n = 4). Fig. 4 View largeDownload slide Subunit molecular ratio of SHMT, TrmFO and ThyX in living cells. (A) To prepare the polyclonal antibodies, SHMT, TrmFO and ThyX were purified and then analysed by 15% SDS- PAGE. The gel was stained with Coomassie Brilliant Blue. (B) The cell extracts were prepared from the wild-type strain cells at early-log (lane 1), mid-log (lanes 2 and 3), and late-log (lane 4) phases. The optical densities of cells at the points are shown at the bottom of panel F. 10 μg of proteins in each sample were analysed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (C) The concentrations of SHMT in the cell extracts were estimated by western blotting analysis. Lanes in the right side of the gel show the bands of standard (purified) SHMT to estimate the quantity. The band intensities were monitored by the fluorescence of secondary antibody (Alexa Fluor 488 anti-rabbit IgG). The amounts of TrmFO (D) and ThyX (E) were estimated by the same method. (F) The results of experiments are summarized. The relative abundances (subunit molecular ratios), which are indicated in the right side, are calculated from the band intensities: the abundance of TrmFO in the extract of mid-log phase cells (optical density at 600 nm is 0.6) is expressed as 1.00 (n = 4). In vitro competition between TrmFO and ThyX via CH2THF consumption We tested in vitro the relationship between TrmFO, ThyX and SHMT. For the measurement of ThyX activity, we adapted the TrmFO assay system (27) allowing analysis of 14C-incorporation into dTMP by thin-layer chromatography (Fig. 5A). In the absence of ThyX, 14C-CH2THF was synthesized by SHMT from 14C-labe1led serine: 14C-labelled serine was converted into 14C-labelled glycine by this reaction (Fig. 5A, left panel). Upon addition of ThyX, 14C-labelled dTMP was synthesized. To estimate the competitive effect of ThyX, we determined TrmFO activity under various concentrations of ThyX and SHMT while keeping the concentrations of TrmFO and tRNAPhe constant (Fig. 5B). Under mimicked natural conditions, with the ratio of SHMT, TrmFO and ThyX being 5:1:1.5, the labelling activity of TrmFO was about 40% (open square indicated by an arrow in Fig. 5B) of that in the absence of ThyX. The presence of ThyX interferes with, but does not completely abrogate TrmFO activity through the consumption of CH2THF (Fig. 5C). This result is in line with the observation that the m5U54 modification was near-fully introduced into the tRNA fraction even under the nutrient-poor condition (Fig. 2C). Because the concentrations of precursor tRNA and dUMP in living cells were unknown, we investigated the ThyX activities in the presence of various concentrations of dUMP and unmodified tRNA (isolated from the ΔtrmFO strain) (Fig. 5D). In the absence of tRNA, the ThyX activity was not dependent on the concentration of dUMP, showing that 7 μM dUMP in the mixture was sufficient for saturated dTMP synthesis (Fig. 5D). After increasing amounts of tRNA were added, the ThyX activity reduced because of competition for CH2THF by TrmFO (Fig. 5D). These in vitro experimental results confirmed that in vivo TrmFO and ThyX are dependent on the concentration of CH2THF, which, in a nutrient-poor environment, determines the speed of DNA-synthesis and thereby that of growth at 70°C. Fig. 5 View largeDownload slide TrmFO and ThyX interfere with each other through competition for CH2THF in vitro. (A) Thin layer chromatography assaying ThyX activity. In the absence of ThyX, SHMT synthesized 14C-labelled CH2THF from 14C-labelled serine (left panel). In the presence of ThyX, dUMP was methylated to 14C-labelled dTMP, which appeared to drive the conversion of 14C-labelled serine by SHMT (right panel). (B) The activity of TrmFO was reduced in the presence of various concentrations of ThyX and SHMT as measured by 3H-methyl group incorporation into unmodified tRNA (isolated from the ΔtrmFO strain) (n = 4). The arrow indicates ThyX activity when the subunit molecular ratio of SHMT, TrmFO and ThyX was 5:1:1.5, mimicking the in vivo situation. The concentration of TrmFO was 0.25 μM. (C) TrmFO activity at various concentrations of ThyX. Under the tested conditions, the presence of a surplus of ThyX did not out-compete TrmFO (0.25 μM) completely. In this experiment, 3H-labelled serine was used. (D) ThyX activity, measured as initial velocities of dTMP formation, under various concentrations of dUMP and tRNA (n = 4). The reaction mixture contained 1.25 μM SHMT, 0.25 μM TrmFO, 0.375 μM ThyX, 5 μM THF, 5 mM NADPH, 25 μM 14C-labelled serine, 0, 0.83, 7.5 or 25 μM tRNAPhe transcript, and 7, 35 or 170 μM dUMP. Fig. 5 View largeDownload slide TrmFO and ThyX interfere with each other through competition for CH2THF in vitro. (A) Thin layer chromatography assaying ThyX activity. In the absence of ThyX, SHMT synthesized 14C-labelled CH2THF from 14C-labelled serine (left panel). In the presence of ThyX, dUMP was methylated to 14C-labelled dTMP, which appeared to drive the conversion of 14C-labelled serine by SHMT (right panel). (B) The activity of TrmFO was reduced in the presence of various concentrations of ThyX and SHMT as measured by 3H-methyl group incorporation into unmodified tRNA (isolated from the ΔtrmFO strain) (n = 4). The arrow indicates ThyX activity when the subunit molecular ratio of SHMT, TrmFO and ThyX was 5:1:1.5, mimicking the in vivo situation. The concentration of TrmFO was 0.25 μM. (C) TrmFO activity at various concentrations of ThyX. Under the tested conditions, the presence of a surplus of ThyX did not out-compete TrmFO (0.25 μM) completely. In this experiment, 3H-labelled serine was used. (D) ThyX activity, measured as initial velocities of dTMP formation, under various concentrations of dUMP and tRNA (n = 4). The reaction mixture contained 1.25 μM SHMT, 0.25 μM TrmFO, 0.375 μM ThyX, 5 μM THF, 5 mM NADPH, 25 μM 14C-labelled serine, 0, 0.83, 7.5 or 25 μM tRNAPhe transcript, and 7, 35 or 170 μM dUMP. Methionine requirement under the nutrient-poor condition As shown in Fig. 2C, several tRNA modifications such as m1A, Cm, m7G, Gm, m1G and m2G were normally introduced into the tRNA fraction under the nutrient-poor condition. The limited nutrients were preferentially consumed in the tRNA modification systems. These methylated nucleosides are conferred by AdoMet-dependent tRNA methyltransferases (2). Thus, this observation suggested that AdoMet at least required for the tRNA methyltransferases was synthesized in the wild-type cells under the nutrient-poor condition. Considering the Km values of tRNA methyltransferases for AdoMet (52–54), at least more than 10 μM of AdoMet seemed to be required in the cells. Because methionine, a precursor of AdoMet, was not added to the minimal synthetic medium, methionine should be synthesized in the cells. Furthermore, methionine is required not only for AdoMet synthesis but also for many biological phenomena such as protein and phospholipid syntheses. In living cells, methionine is synthesized by methionine synthase (MetE) from homocysteine and N5-methyltetrahydoxylfolate (5-methylTHF), which is synthesized from CH2THF by MetF (55). Thus, considerable amount of CH2THF seemed to be consumed for the methionine synthesis under a nutrient-poor condition. To confirm this idea, we added methionine to the minimal synthetic medium (Supplementary Fig. S3). In the absence of methionine, the ΔtrmFO strain grew faster than the wild-type strain (Fig. 2A). When 100 or 300 μM of methionine was added to the medium, slight acceleration of growth of the ΔtrmFO strain was observed (data not shown). In contrast, when methionine was added to 1 mM, the wild-type and ΔtrmFO strains grew at almost the same speed (Supplementary Fig. S3). This result showed that the consumption of CH2THF in the methionine synthesis is a very important factor to decide the growth speed under the nutrient-poor condition. Because the addition of thymidine did not cause perfect recovery of growth speed (Fig. 3B), we concluded that the main reason of slow growth of the wild-type strain was the consumption of CH2THF for the methionine synthesis. Activities of TrmFO and MetF compete in in vitro experiments In T. thermophilus cells, methionine is synthesized from homocysteine and 5-methylTHF, which is converted from CH2THF by the reaction of MetF (56). Therefore, we tested whether the activity of MetF competes with the activity of TrmFO. It should be mentioned that T. thermophilus has the simplest MetF system (50, 56, 57): T. thermophilus MetF did not require other proteins for the activity and has only CH2THF-dependent NADH oxidase activity. T. thermophilus MetF was expressed in E. coli cells and purified (Fig. 6A). We confirmed the NADH oxidase activity. In brief, 0.4 μM SHMT, 5 μM THF, 25 μM serine and 200 μM NADH were incubated at 20°C for 10 min and then 3.0 μM MetF was added into the reaction mixture. As shown in Fig. 6B, NADH was consumed by the addition of MetF. This reaction was checked by formation of 14C-5-methylTHF from 14C-CH2THF using 14C-labelled serine (Fig. 6C). After these pilot experiments, TrmFO activities were measured at 60°C in the absence and presence of MetF (Fig. 6D). The activity of TrmFO was decreased in the presence of MetF. Thus, the activity of MetF competes with the activity of TrmFO in vitro. However, even in the presence of 10-fold larger amount of MetF, the methylation by TrmFO was still observed (bottom panels). This observation may explain the near-complete formation of m5U54 in tRNA under the nutrient-poor condition. In living cells, 5-methylTHF produced by MetF is converted to methionine by the reaction of MetE. Therefore, under the nutrient-poor condition, CH2THF may be consumed by the reactions of MetF and MetE more rapidly as compared with the results of in vitro experiments. Fig. 6 View largeDownload slide Activity of MetF competes with activity of TrmFO. (A) 8 μg of purified MetF was analysed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (B) NADH oxidase activity of MetF was monitored by decrease of absorbance at 340 nm. 0.4 μM SHMT, 5 μM THF, 25 μM serine and 200 μM NADH were incubated at 20°C for 10 min and then 3.0 μM MetF was added to the reaction mixture. (C) The formation of 5-methylTHF by MetF was checked using 14C-labelled serine. 14C-labelled CH2THF was synthesized by the activity of SHMT. The reaction was started by the addition of 14C-labelled serine. After 10 min, 3.0 μM MetF was added to the reaction mixture. (D) The activities of TrmFO in the absence and presence of MetF were analysed by 14C-methyl group incorporations into tRNAPhe transcript. The subunit molecular ratios of TrmFO and MetF are indicated at the left side. The gels were stained with methylene blue (left panels) and their autoradiograms were taken (right panels). Fig. 6 View largeDownload slide Activity of MetF competes with activity of TrmFO. (A) 8 μg of purified MetF was analysed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (B) NADH oxidase activity of MetF was monitored by decrease of absorbance at 340 nm. 0.4 μM SHMT, 5 μM THF, 25 μM serine and 200 μM NADH were incubated at 20°C for 10 min and then 3.0 μM MetF was added to the reaction mixture. (C) The formation of 5-methylTHF by MetF was checked using 14C-labelled serine. 14C-labelled CH2THF was synthesized by the activity of SHMT. The reaction was started by the addition of 14C-labelled serine. After 10 min, 3.0 μM MetF was added to the reaction mixture. (D) The activities of TrmFO in the absence and presence of MetF were analysed by 14C-methyl group incorporations into tRNAPhe transcript. The subunit molecular ratios of TrmFO and MetF are indicated at the left side. The gels were stained with methylene blue (left panels) and their autoradiograms were taken (right panels). Adenine requirement under the nutrient-poor condition CH2THF is used for purine synthesis in addition to tRNA methylation, dTMP synthesis and methionine synthesis. To investigate whether purine is an important factor for growth speed under the nutrient-poor condition, we added 100 μM of adenine into the synthetic medium. As shown in Supplementary Fig. S4, the growth speed of the wild-type strain was not changed. Thus, purine is not a rate-limiting factor of growth under the nutrient-poor condition. This result suggests that purine is supplied from the salvage system under the tested condition. Discussion The ΔtrmFO strain can grow faster than the wild-type strain in the synthetic medium at 70°C. As far as the authors know, until now, there is no report that a gene disruption of tRNA modification enzyme accelerates the growth. At the beginning of study, we assumed abnormal (or insufficient) modification(s) in tRNA. In the wild-type cells, TrmFO binds to precursor tRNA and then methylates U54. We supposed that this binding might have effect on the binding of other tRNA modification enzymes. However, contrary to our expectation, we could not find any abnormal modification in the tRNA fraction from the ΔtrmFO strain that was cultured under the nutrient-poor condition. The rapid growth of ΔtrmFO strain was observed only under the nutrient-poor condition: the wild-type and ΔtrmFO strains grow at the same speed in the nutrient-rich medium at 70°C (36). This observation prompted us to investigate the CH2THF consumption. The results of this study are summarized in Fig. 7. In the wild-type strain, CH2THF is mainly consumed by two metabolic pathways, dTMP and methionine syntheses. The presence of TrmFO decreases the speeds of dTMP and methionine syntheses. This is the reason of slow growth of the wild-type strain at 70°C under the nutrient-poor condition. Our in vitro experimental results revealed that TrmFO and ThyX, and TrmFO and MetF compete at several enzyme concentrations. The methionine synthesis pathway is complicatedly regulated: the expression levels of enzymes in the methionine metabolism are regulated by the concentrations of methionine and AdoMet (55). Therefore, in the case of methionine synthesis, our results of in vitro experiments may be not directly applied to in vivo phenomena. However it is clear that considerable amount of AdoMet is synthesized via methionine under the nutrient-poor condition because the majority of methylated nucleosides were normally introduced into the tRNA fraction. In contrast, in the case of ΔtrmFO strain, there is no consumption of CH2THF by TrmFO. Thus, the limited amount of CH2THF can be used preferentially for main pathways, and this advantage results in the rapid growth. Fig. 7 View largeDownload slide Consumption of CH2THF in T. thermophilus under the nutrient-poor condition at 70°C. Under the nutrient-poor condition, CH2THF is consumed by these pathways as described in the main text. In the case of ΔtrmFO strain, there is no consumption of CH2THF by TrmFO. Fig. 7 View largeDownload slide Consumption of CH2THF in T. thermophilus under the nutrient-poor condition at 70°C. Under the nutrient-poor condition, CH2THF is consumed by these pathways as described in the main text. In the case of ΔtrmFO strain, there is no consumption of CH2THF by TrmFO. T. thermophilus lives in hot springs, in which nutrients such as amino acids are limited like in the nutrient-poor condition of our tests. Our experimental results revealed that the limited nutrients in the nutrient-poor condition are preferentially consumed in the tRNA modification systems. Modified nucleosides in tRNA are required for accurate and efficient protein synthesis (2, 25). If the tRNA modifications are insufficient, it directly causes the defects in the protein synthesis. For example, the absence of m1G37 modification in tRNA causes the frame-shift error (58) and incorrect recognition by aminoacyl-tRNA synthetase (59). Furthermore, mnm5U derivatives at position 34 are required for correct reading of codons and maintenance of reading frame (60–64). The errors in protein synthesis abolish numerous ATP, GTP and amino acids. Therefore, the modifications in tRNA may be preferentially introduced. The m5U54 modification by TrmFO is not essential for viability of T. thermophilus and is required only for the assistant of Ψ55 modification at low temperatures. T. thermophilus would be exposed to a wide range of temperatures, for example when hot spring water is cooled down by influx of river water or snow fall, while the hot spring can flow over or experience an eruption of hot water. The fine tuning of protein synthesis through the network of modified nucleosides in tRNA and tRNA modification enzymes seems to be very important for the survival of T. thermophilus. The physiological reason why TrmFO uses CH2THF is unclear. The m5U54 itself can be synthesized from AdoMet. Indeed, several eubacteria including E. coli possess AdoMet-dependent tRNA (m5U54) methyltransferase (TrmA) (13). Because AdoMet is synthesized by multiple steps from CH2THF under the nutrient-poor condition, consumption of AdoMet seems to be in the same situation as that of CH2THF from the viewpoint of one carbon metabolism. AdoMet is consumed in many metabolic pathways such as lipid synthesis, DNA methylation and polyamine synthesis in addition to the RNA modification. T. thermophilus produces various polyamines including long and branched polyamines (65). Two proteins, triamine/agmatine aminopropyltransferase (SpeE) and S-adenosyl-l-methionine decarboxylase-like protein 1 (SpeD1), are required for biosynthesis of long and branched polyamines from spermidine (66, 67). The gene-deletion strains of speE and speD1 could not grow in the synthetic medium at above 75°C (67). In contrast, the wild-type strain can grow at 80°C under the nutrient-poor condition. These observations suggest that considerable amount of AdoMet is consumed in the biosynthetic pathway of polyamines under the nutrient-poor condition. The rapid growth of ΔtrmFO strain under the nutrient-poor condition was only observed at 70°C. At 50°C, the wild-type and ΔtrmFO strains grew at nearly the same speed (data not shown). These observations suggest that the rate-limiting factor(s) for growth speed may be changed according to the culture temperatures. At the optimal temperature (70°C), the speed of dTMP and methionine syntheses seem to have the direct effect on the growth speed. Finally, this study revealed that tRNA modification systems in T. thermophilus preferentially consume the limited nutrients (CH2THF and AdoMet) under the nutrient-poor condition. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements We thank previous collaborators (Dr Chie Tomikawa in Ehime University, Japan and Prof Satoko Yoshizawa in CNRS, France) for valuable discussion. Funding This work was supported by a Grant-in-Aid for JSPS Fellows 26-8015(to R.Y.), a Grant-in-Aid for Scientific Researches 23350081 and 16H04763 (to H.H.) and a Grant-in-Aid for Exploratory Research 24655156 (to H.H.) from the Japan Society for the Promotion of Science (JSPS). Conflict of Interest None declared. 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( 2017) Long and branched polyamines are required for maintenance of the ribosome, tRNAHis and tRNATyr in Thermus thermophilus cells at high temperatures. Genes Cells  22, 628– 645 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations AdoMet S-adenosyl-l-methionine CH2THF N5, N10-methylenetetrahydrofolate cmnm5U 5-carboxymethylaminomethyluridine D dihydrouridine Gm 2′-O-methylguanosine m1A 1-methyladenosine 5-methylTHF N5-methyltetrahydroxylfolate m7G 7-methylguanosine MnmEG MnmE and MnmG complex mnm5U 5-methylaminomethyluridine m5s2U 5-methyl-2-thiouridine m5U 5-methyluridine PAGE polyacrylamide gel electrophoresis Ψ pseudouridine THF tetrahydrofolate © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journal of Biochemistry – Oxford University Press

Published: Mar 10, 2018

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