Reversible RNA phosphorylation stabilizes tRNA for cellular thermotolerance

Archaeal strains and media

S. tokodaii str. 7, Methanosarcina acetivorans C2A and Thermoplasma acidophilum were kindly provided by T. Oshima (Kyowa Kako Co., Ltd), T. Yokogawa (Gifu University) and H. Hori (Ehime University), respectively. Sulfolobus acidocaldarius (JCM no. 8929), Saccharolobus solfataricus (JCM no. 8930), Aeropyrum pernix (JCM no. 9820), Pyrobaculum oguniense (JCM no. 10595) and N. viennensis (JCM no. 19564) were obtained from Japan Collection of Microorganisms, RIKEN BRC which is participating in the National BioResource Project of the MEXT, Japan.

S. tokodaii and S. acidocaldarius were cultured at 80 °C in JCM medium no. 165 consisting of 1 g l–1 yeast extract, 1 g l–1 casamino acids, 1.3 g l–1 (NH4)2SO4, 0.28 g l–1 KH2PO4, 0.25 g l–1 MgSO4·7H2O, 0.07 g l–1 CaCl2·2H2O, 2.0 mg l–1 FeCl3·6H2O, 1.8 mg l–1 MnCl2·4H2O, 4.5 mg l–1 Na2B4O7·10H2O, 0.22 mg l–1 ZnSO4·7H2O, 0.05 mg l–1 CuCl2·2H2O, 0.03 mg l–1 Na2MoO4·2H2O, 0.03 mg l–1 VOSO4·H2O and 0.01 mg l–1 CoSO4·7H2O (adjusted to pH 2.5 with H2SO4). S. solfataricus was cultured at 80 °C in JCM medium no. 171 consisting of 1 g l–1 yeast extract, 2.5 g l–1 (NH4)2SO4, 3.1 g l–1 KH2PO4, 0.2 g l–1 MgSO4·7H2O, 0.25 g l–1 CaCl2·2H2O, 1.8 mg l–1 MnCl2·4H2O, 4.5 mg l–1 Na2B4O7·10H2O, 0.22 mg l–1 ZnSO4·7H2O, 0.05 mg l–1 CuCl2·2H2O, 0.03 mg l–1 Na2MoO4·2H2O, 0.03 mg l–1 VOSO4·H2O and 0.01 mg l–1 CoSO4·7H2O (adjusted to pH 4.0 with H2SO4). A. pernix was cultured at 90 °C in JCM medium no. 224 consisting of 1 g l–1 yeast extract, 1 g l–1 peptone, 1 g l–1 Na2S2O3·5H2O, 24.0 g l–1 NaCl, 7.0 g l–1 MgSO4·7H2O, 5.3 g l–1 MgCl2·6H2O, 0.7 g l–1 KCl and 0.1 g l–1 CaCl2·2H2O (adjusted to pH 7.0 with NaOH). P. oguniense was cultured at 90 °C in JCM medium no. 165 with addition of 1.0 g l–1 Na2S2O3·5H2O (adjusted to pH 7.25 with NaOH). N. viennensis was cultured at 42 °C in JCM medium no. 1004 consisting of 1 g l–1 NaCl, 0.5 g l–1 KCl, 0.4 g l–1 MgCl2·6H2O, 0.2 g l–1 KH2PO4, 0.1 g l–1 CaCl2·2H2O, 1.0 ml l–1 modified trace element mixture (30 mg l–1 H3BO3, 100 mg l–1 MnCl2·4H2O, 190 mg l–1 CoCl2·6H2O, 24 mg l–1 NiCl2·6H2O, 2 mg l–1 CuCl2·2H2O, 144 mg l–1 ZnSO4·7H2O, 36 mg l–1 Na2MoO4·2H2O and 0.3% HCl), 1.0 ml l–1 vitamin solution (20 mg l–1 biotin, 20 mg l–1 folic acid, 100 mg l–1 pyridoxine·HCl, 50 mg l–1 thiamine·HCl, 50 mg l–1 riboflavin, 50 mg l–1 nicotinic acid, 50 mg l–1 DL-calcium pantothenate, 1 mg l–1 vitamin B12, 50 mg l–1 p-aminobenzoic acid and 2 g l–1 choline chloride (adjusted to pH 7.0 with KOH)), 1.0 ml l–1 7.5 mM EDTA·Na·Fe(III) solution (pH 7.0), 2.0 ml l–1 1 M NaHCO3 solution, 10 ml l–1 HEPES solution (238.4 g l–1 HEPES (free acid) and 24 g l–1 NaOH), 1.0 ml l–1 1 M NH4Cl solution and 1.0 ml l–1 1 M sodium pyruvate solution (adjusted to pH 7.6 with NaOH).

T. kodakarensis was cultured at 83 °C, 87 °C or 91 °C, in nutrient-rich medium (ASW-YT-S0 or MA-YT-Pyr) or synthetic medium containing amino acids (ASW-AA-S0), under strict anaerobic conditions. ASW-YT-S0 medium contains 0.8× artificial sea water (ASW)50 , 10 g l–1 yeast extract, 5.0 g l–1 tryptone, 2.0 g l–1 elemental sulfur and 0.1% (wt/vol) resazurin. MA-YT-Pyr medium contains 30.5 g l–1 Marine Art SF-1 (Osaka Yakken), 10 g l–1 yeast extract, 5.0 g l–1 tryptone, 5.0 g l–1 pyruvate sodium and 0.1% (wt/vol) resazurin. ASW-AA-S0 medium contains 0.8× ASW, 0.5× amino acid solution50, modified Wolfe’s trace minerals (0.5 g l–1 MnSO4·2H2O, 0.1 g l–1 CoCl2, 0.1 g l–1 ZnSO4, 0.01 g l–1 CuSO4·5H2O, 0.01 g l–1 AlK(SO4)2, 0.01 g l–1 H3BO3 and 0.01 g l–1 NaMoO4·2H2O), 5.0 ml l–1 vitamin mixture51, 2.0 g l–1 elemental sulfur and 0.1% (wt/vol) resazurin. For plate cultivation, 2.0 ml l–1 polysulfide solution (20% elemental sulfur in 67% Na2S·9H2O solution) was added instead of elemental sulfur, and the media were solidified with 1.0% Gelrite (Fujifilm Wako Pure Chemical Corporation). When pyrF-negative transformants were selected0, 75% 5-fluoroorotic acid (5-FOA) was added. We used ASW-YT-S0 medium for standard cultivation, MA-YT-Pyr medium for growth comparisons and ASW-AA-S0 medium for construction of the gene knockout strain.

Preparation of tRNA fractions

For small-scale preparation (~100-ml culture), archaeal cells were resuspended in 3 ml solution D (4 M guanidine thiocyanate, 25 mM citrate–NaOH (pH 7.0), 0.5% (wt/vol) N-lauroylsarcosine sodium salt and 1 mM 2-mercaptoethanol) and mixed with an equal volume of water-saturated phenol and 1/10 volume of 3 M sodium acetate (pH 5.3). The mixture was shaken for 1 h on ice and mixed with 1/5 volume of chloroform, followed by centrifugation at 8,000g for 10 min at 4 °C. The supernatant was collected and mixed with an equal volume of chloroform, followed by centrifugation at 8,000g for 10 min at 4 °C. Total RNA was obtained from the resultant supernatant by isopropanol precipitation. The total RNA prepared in this manner was separated by 10% denaturing PAGE, followed by staining with SYBR Gold or toluidine blue. The visualized tRNA fraction including class I and class II tRNAs was cut out and eluted from the gel slice with elution buffer (0.3 M sodium acetate (pH 5.3) and 0.1% (wt/vol) SDS), followed by filtration to remove the gel pieces and ethanol precipitation for RNA-MS analysis of the tRNA fraction.

For large-scale preparation of tRNA fractions from S. tokodaii, cell pellets (53 g) were resuspended in 530 ml solution D and then mixed with 53 ml of 3 M sodium acetate (pH 5.3) and 425 ml neutralized phenol. The mixture was shaken for 1 h on ice to which 106 ml chloroform/isoamyl alcohol (49:1) was added, followed by centrifugation at 4,500g for 20 min at 4 °C. The supernatant was collected and mixed with 106 ml chloroform/isoamyl alcohol (49:1), followed by centrifugation at 4,500g for 15 min at 4 °C. The aqueous phase was collected and then subjected to isopropanol precipitation. The collected RNA was resuspended in 53 ml water and mixed with 80 ml TriPure Isolation Reagent (Roche), followed by centrifugation at 10,000g for 20 min at 4 °C. The supernatant was collected and mixed with 36 ml chloroform/isoamyl alcohol (49:1), followed by centrifugation at 10,000g for 10 min at 4 °C. The aqueous phase was collected and precipitated with isopropanol. The prepared total RNA (608 mg) was dissolved in 250 ml of buffer consisting of 20 mM HEPES-KOH (pH 7.6), 200 mM NaCl and 1 mM DTT and then loaded on a DEAE Sepharose Fast Flow column (320-ml beads) and fractionated with a gradient of NaCl from 200 to 500 mM. Fractions containing tRNA were collected by isopropanol precipitation.

Isolation of individual tRNAs

Isolation of individual tRNAs from thermophilic organisms is extremely difficult owing to their high melting temperatures, which are the consequence of their high G+C content and complex modifications. We thus optimized our original method for RNA isolation by RCC24 or chaplet column chromatography (CCC)52. Approximately 200 absorbance at 260 nm (A260) units of the S. tokodaii tRNA fraction was subjected to RCC. The isolation procedure was carried out as follows: hybridization at 66 °C in 6× NHE buffer (30 mM HEPES-KOH (pH 7.5), 15 mM EDTA (pH 8.0), 1.2 M NaCl, 1 mM DTT), washing at 50 °C with 0.1× NHE buffer (0.5 mM HEPES-KOH (pH 7.5), 0.25 mM EDTA (pH 8.0), 20 mM NaCl, 0.5 mM DTT) and elution at 72 °C with 0.1× NHE buffer. Eluted tRNAs were recovered by ethanol precipitation. Mature and precursor tRNAs were separated by 10% denaturing PAGE and stained with SYBR Gold. Visualized bands of mature and precursor tRNAs were cut out and eluted from the gel slices with elution buffer, followed by filtration to remove the gel pieces and precipitation with ethanol.

To crystalize native tRNA bearing Up47, we conducted large-scale isolation of S. tokodaii tRNAVal3 using CCC52. The S. tokodaii tRNA fraction (2,000 A260 units) was subjected to CCC with tandem affinity chaplet columns for tRNAVal3, tRNAIle2 and tRNAPhe. The isolation procedure was carried out as follows: hybridization at 66 °C in 6× NHE buffer, washing separately at 50 °C with 0.1× NHE buffer and elution at 72 °C with 0.1× NHE buffer. The eluted tRNAs were recovered by isopropanol precipitation. The sequences of the DNA probes are shown in Supplementary Table 6. The isolated tRNAVal3 was further purified by anion exchange chromatography to completely remove tRNAVal2, as described below.

RNA mass spectrometry

For tRNA fragment analysis by RNA-MS, 30 ng (900 fmol) of the isolated tRNA or 150 ng (4.5 pmol) of tRNA mixture was digested with RNase T1 (Epicentre or Thermo Fisher Scientific) or RNase A (Ambion) and analysed with a linear ion trap–Orbitrap hybrid mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific) equipped with a custom-made nanospray ion source and a splitless nanoHPLC system (DiNa, KYA Technologies) as described previously26,27. To analyse Ψ sites, tRNA was treated with acrylonitrile to cyanoethylate Ψ53 and subjected to RNA-MS. For dephosphorylation of the Up47-containing fragment (Extended Data Fig. 4a, b), RNase T1 digestion was performed in the presence of 0.01 U μl–1 bacterial alkaline phosphatase (BAP C75, Takara Bio). To precisely map tRNA modifications, RNA fragments were decomposed by CID in the instrument. The normalized collision energy of LTQ Orbitrap XL was set to 40%. Mongo Oligo Mass Calculator v2.08 (https://mods.rna.albany.edu/masspec/Mongo-Oligo) was used for assignment of the product ions in CID spectra.

For nucleoside analysis, 800 ng (24 pmol) of the isolated tRNAVal3 was digested with 0.09 U nuclease P1 (Fujifilm Wako Pure Chemical Corporation) in 20 mM ammonium acetate (pH 5.2) at 50 °C for 1 h and mixed with 1/8 volume of 1 M trimethylamine-HCl (TMA-HCl) (pH 7.2) and 0.06 U phosphodiesterase I (Worthington Biochemical Corporation), followed by incubation at 37 °C for 1 h. To this mixture, 0.08 U BAP was added, and the sample was incubated at 50 °C for 1 h. After that, 9 volumes of acetonitrile were added, followed by LC–MS/MS analysis as described in refs. 25,54 with some modifications as follows. The samples were chromatographed with a ZIC-cHILIC column (3-μm particle size, 2.1 × 150 mm; Merck) and eluted with 5 mM ammonium acetate (pH 5.3) (solvent A) and acetonitrile (solvent B) at a flow rate of 100 μl min–1 with a multistep linear gradient: 90–50% solvent B for 30 min, 50% solvent B for 10 min, 50–90% solvent B for 5 min and then initialization with 90% solvent B. The chromatographed eluent was directly introduced into the electrospray ionization source of the Q Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Fisher Scientific).

For nucleotide analysis, 800 ng (24 pmol) of the tRNA fraction or individual tRNA was digested with 0.09 U nuclease P1 in 20 mM ammonium acetate (pH 5.2) at 50 °C for 1 h and then mixed with 9 volumes of acetonitrile for LC–MS. The digests were chromatographed with a ZIC-cHILIC column and analysed by Q Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Fisher Scientific) or LTQ Orbitrap XL (Thermo Fisher Scientific) with a multistep linear gradient: 90–50% solvent B for 30 min, 50% solvent B for 10 min, 50–90% solvent B for 5 min and then initialization with 90% solvent B.

The acquired LC–MS data were analysed using Xcalibur 4.1 (Thermo Fisher Scientific) and were visualized with Canvas X (Nihon poladigital k.k).

Isolation and detection of pN324p

Five A260 units of the S. tokodaii tRNA fraction was completely digested with nuclease P1. Digests containing pN324m5C dinucleotide were subjected to periodate oxidation with 10 mM NaIO4 for 1 h on ice in the dark. The reaction was stopped by addition of 1 M l-rhamnose and incubation for 30 min. For β-elimination, an equal volume of 2 M lysine-HCl (pH 8.5) was added, and the sample was incubated at 45 °C for 90 min. The product containing pN324p was then subjected to anion exchange chromatography with a Q Sepharose Fast Flow column (GE Healthcare) equilibrated with 20 mM triethylammonium bicarbonate (TEAB) (pH 8.2). The eluate with 2 M TEAB was collected and dried by evaporation in vacuo. The pellet was dissolved with water and mixed with an equal volume of chloroform, followed by centrifugation at 20,000g for 5 min at 4 °C. The supernatant was recovered and dried again. This process was repeated five times. The resultant digest was mixed with 9 volumes of acetonitrile and subjected to LC–MS/MS using an LCQ-Advantage ion trap mass spectrometer (Thermo Scientific), equipped with an electrospray ionization source and an HP1100 LC system (Agilent Technologies). For LC, the digest was chromatographed with a ZIC-HILIC column (3.5 μm; pore size, 100 Å; internal diameter, 2.1 × 150 mm; Merck) and eluted with 5 mM formic acid (pH 3.4) (solvent A) and acetonitrile (solvent B) at a flow rate of 100 μl min–1 with a multistep gradient: 90–70% solvent B for 25 min, 70–10% solvent B for 15 min, 10% solvent B for 5 min and then initialized with 90% solvent B.

Expression and purification of recombinant proteins

Synthetic genes for arkI from T. kodakarensis, Methanocaldococcus fervens, P. oguniense, Aquifex aeolicus, Nautilia profundicola and Leptolyngbya sp. PCC7376 were designed with codons optimized for E. coli expression and synthesized by GENEWIZ or Thermo Fisher Scientific. Each gene was cloned into the pE-SUMO-TEV vector by the SLiCE method55. N. viennensis arkI was PCR amplified from genomic DNA with a set of primers (Supplementary Table 6) and cloned into the BamHI and NotI sites of pE-SUMO-TEV.

E. coli BL21(DE3) or Rosetta2(DE3) cells transformed with the pE-SUMO-TEV vector carrying each arkI gene were cultured in 250 ml or 1 l of LB containing 50 μg ml–1 kanamycin and 20 μg ml–1 chloramphenicol when necessary. His6–SUMO-tagged recombinant protein was expressed at 37 °C for 3–4 h by induction with 0.1 or 1 mM IPTG or 2% (wt/vol) lactose when the cells reached OD610 = 0.4–0.6. P. oguniense ArkI was expressed in cells cultured overnight at 18 °C. The collected cells were resuspended in lysis buffer (50 mM HEPES-KOH (pH 8.0), 150 mM KCl, 2 mM MgCl2, 20 mM imidazole, 12% (vol/vol) glycerol, 1 mM 2-mercaptoethanol and 1 mM PMSF) and disrupted by sonication, followed by centrifugation at 15,000g for 15 min at 4 °C. The supernatant was boiled at 60 °C for 20 min (for ArkI homologues from T. kodakarensis, M. fervens, P. oguniense and A. aeolicus) and centrifuged at 15,000g for 15 min at 4 °C. The recombinant protein was affinity captured on an Ni-Sepharose 6 Fast Flow column (GE Healthcare) and then eluted with lysis buffer containing 300 mM imidazole, followed by gel filtration with a PD-10 column (GE Healthcare) to remove the imidazole. The recombinant protein for N. viennensis ArkI was purified using a HisTrap column (GE Healthcare) with a linear gradient of 0–500 mM imidazole, followed by dialysis using a Slide-A-Lyzer Dialysis Cassette (Thermo Fisher Scientific) to remove imidazole. The purified protein was subjected to Ulp1 digestion at 4 °C overnight to cleave the His6–SUMO tag and then passed through a Ni-Sepharose 6 Fast Flow column to remove the tag. Because ArkI homologues from M. fervens (MfArkI) and Leptolyngbya sp. PCC7376 (LeArkI) aggregated following tag removal, His6–SUMO tag-fused proteins of these homologues were used for the phosphorylation assay. Purified protein was quantified by the Bradford method using BSA as a standard.

For large-scale preparation of T. kodakarensis ArkI for crystallization, the E. coli BL21(DE3) strain carrying pE-SUMO-TkArkI was cultured in 2 l of LB containing 50 μg ml–1 kanamycin and TkArkI was expressed at 25 °C overnight by induction with 0.1 mM IPTG when the cells reached OD610 = 0.4. The cells were collected and disrupted by sonication in lysis buffer (50 mM HEPES-KOH (pH 8.0), 150 mM KCl, 2 mM MgCl2, 20 mM imidazole, 12% (vol/vol) glycerol, 1 mM 2-mercaptoethanol and 1 mM PMSF). The protein was purified using a HisTrap column with a linear gradient of 20–520 mM imidazole. Fractions containing TkArkI were pooled and subjected to Ulp1 digestion at 4 °C overnight to cleave the tag, followed by passage through a Ni-Sepharose 6 Fast Flow column to remove the tag fragment. The flow-through fraction was filtered through a 0.45-μm PVDF membrane to remove the resin. The protein was further purified by affinity chromatography with a HiTrap Heparin HP column (GE Healthcare) using a linear gradient of 150–1,150 mM KCl. TkArkI was further purified by size exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare) with buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10 mM 2-mercaptoethanol and then concentrated to 5.74 mg ml–1 and stored at –80 °C.

The T. kodakarensis kptA gene was PCR amplified from genomic DNA from T. kodakarensis with the primers listed in Supplementary Table 6 and cloned into pE-SUMO-TEV to give pE-SUMO-TEV-tkkptA. The E. coli Rosetta2(DE3) strain carrying pE-SUMO-TEV-tkkptA was cultured in 1 l LB containing 50 μg ml–1 kanamycin and 20 μg ml–1 chloramphenicol, and TkKptA was expressed at 37 °C for 3 h by induction with 0.1 mM IPTG when the cells reached OD610 = 0.6. The recombinant TkKptA was purified as described above. The gene encoding Tpt1p was PCR amplified from the genomic DNA of S. cerevisiae BY4742 with the set of primers listed in Supplementary Table 6 and was cloned into pET21b (Merck) between the NdeI and XhoI sites. Recombinant Tpt1p was purified as described above.

Removal of the 2′-phosphate of Up47 by Tpt1p

Removal of the 2′-phosphate of Up47 by yeast Tpt1p was performed as described33. Individual tRNAs or the tRNA fraction was incubated for 3 h at 30 °C in a reaction mixture (25 μl) consisting of 20 mM Tris-HCl (pH 7.4), 0.5 mM EDTA (pH 8.0), 1 mM NAD+, 2.5 mM spermidine, 0.1 mM DTT, 0.9 μM tRNA and 0.1 μg μl–1 recombinant Tpt1p. The tRNA was extracted by phenol/chloroform treatment and recovered by ethanol precipitation, followed by desalting with Centri-Sep spin columns (Princeton Separations). For crystallization of Tpt1p-treated tRNA, S. tokodaii tRNAVal3 (202.5 μg) was dephosphorylated by yeast Tpt1p in a 200-μl reaction mixture.

Measurement of the thermal stability of tRNA

S. tokodaii tRNAVal3 (25 pmol) with or without Up47 was dissolved in degassed buffer consisting of 50 mM Tris-HCl (pH 7.4), 100 mM NaCl and 1 mM MgCl2 and incubated at 80 °C for 5 min, followed by cooling to 25 °C at a rate of 0.1 °C s–1. The samples were placed onto a Type 8 multi-micro UV quartz cell (path length, 10 mm). The hyperchromicity of tRNA was monitored on a UV–visible light spectrophotometer (V-630, JASCO). The gradients were as follows: 25 °C for 30 s, 25–40 °C at 5 °C min–1, 40 °C for 5 min and 40–105 °C at 0.5 °C min–1. The Tm was calculated using Spectra Manager v2 (JASCO). Melting curves were generated using Microsoft Excel.

RNase probing of tRNA

S. tokodaii tRNAVal3 (25 pmol) with or without Up47 was labelled with 32P at the 3′ terminus by ligation with [5′-32P]cytidine 3′,5′-bisphosphate (PerkinElmer). The labelled tRNA was separated on a 7.5% (wt/vol) polyacrylamide gel containing 7 M urea, 1× TBE and 10% (vol/vol) glycerol and was purified by gel extraction. Labelled tRNA was mixed with the S. tokodaii tRNA fraction as a carrier to a concentration of 100,000 counts per minute (c.p.m.) per A260 unit and was precipitated with ethanol. The pellet was dissolved in water to a concentration of 0.1 A260 units per μl. For the RNase degradation assay, the labelled tRNA (0.1 A260 units, 10,000 c.p.m.) was incubated at 65 °C in a reaction mixture consisting of 10 mM HEPES-KOH (pH 7.6), 0.5 mM MgCl2, 100 mM NaCl and 0.1 U μl–1 RNase I (Promega). At time points of 1, 3, 5, 10, 15 and 30 min after starting the reaction, aliquots were taken from the mixture and mixed well with chilled phenol/chloroform/isoamyl alcohol (25:24:1, pH 7.9) to stop the reaction, followed by centrifugation at 15,000g for 15 min at 4 °C. The supernatant was collected and treated with an equal volume of chloroform, followed by centrifugation at 15,000g for 5 min at 4 °C. The supernatant was mixed with 2× loading solution (2× TBE, 7 M urea, 13.33% (wt/vol) sucrose, 0.05% (wt/vol) xylene cyanol and 0.05% (wt/vol) bromophenol blue) and subjected to 10% denaturing PAGE. The gel was exposed to an imaging plate, and radioactivity was visualized by using an FLA-7000 imaging analyser (Fujifilm). Graphs were generated using Microsoft Excel.

Crystallization of S. tokodaii tRNAVal3

S. tokodaii tRNAVal3 (500 μg), isolated as described above, was refolded in annealing buffer (50 mM HEPES-KOH (pH 7.6), 5 mM MgCl2 and 1 mM DTT) by incubation for 5 min at 80 °C and cooling to 25 °C with a rate of 0.1 °C s–1. tRNAVal3 was further purified by anion exchange chromatography using a Mono Q 5/50 GL column (GE Healthcare) with a linear gradient of 200–1,000 mM NaCl. The major peak was collected, precipitated with isopropanol, dissolved in water and precipitated with ethanol. Tpt1p-treated tRNAVal3 was prepared with the same procedure as described above. The purified tRNA was dissolved in buffer consisting of 10 mM Tris-HCl (pH 7.1) and 5 mM MgCl2 to a concentration of 50 μM. One microlitre of tRNA solution was mixed with 1 μl Natrix 2 no. 32 (80 mM NaCl, 12 mM spermine-4HCl, 40 mM sodium cacodylate·3H2O (pH 7.0) and 30% (vol/vol) MPD) (Hampton Research) on silicon-coated glass and crystalized by the hanging drop vapor diffusion method at 20 °C.

Crystallization of T. kodakarensis ArkI

The concentration of TkArkI was adjusted to 5 mg ml–1 before crystallization. One microlitre of the protein solution was mixed with 0.5 μl reservoir solution, containing 25% (vol/vol) ethylene glycol. TkArkI was crystallized by the hanging drop vapor diffusion method at 20 °C.

Data collection and crystal structure determination

The datasets were collected at beamline BL-17A at the Photon Factory at KEK, Japan. For data collection for the tRNAVal3 crystals, the crystals were cryoprotected with a portion of the reservoir solution. For data collection for the native TkArkI crystal, the crystal was cryoprotected with solution containing 25% (vol/vol) ethylene glycol, 2 mM MgCl2 and 1 mM ATP. For data collection for the iodide-derivative TkArkI crystal, the crystal was briefly soaked in and cryoprotected with solution containing 300 mM potassium iodide and 22.5% (vol/vol) ethylene glycol, and the diffraction dataset was collected at a wavelength of 1.5 Å. The datasets were indexed, integrated and scaled using xds56. The initial phase of tRNAVal3 was determined by molecular replacement with Phaser57. The structure of T. thermophilus tRNAVal (PDB, 1IVS)58 was used for the model. The initial phase of TkArkI was determined by the SAD method using the anomalous signal of iodide ions. The iodine sites were located by SHELX59, and the initial phase was calculated by Phaser. Subsequent density modification and initial model building were performed with RESOLVE60. The model was further modified with Coot61 and refined with Phenix62. Crystal structures and their electron density maps were visualized using PyMOL, Cuemol or Coot. Torsion angles of the tRNAs were analysed with DSSR software63.

Analysis of ligands bound to TkArkI

TkArkI purified by affinity chromatography with a HiTrap Heparin HP column (GE Healthcare) (100 pmol) was mixed with [15N]adenosine (10 pmol) and [15N]guanosine (10 pmol) as tracer molecules, followed by addition of 4 volumes of methanol, an equal volume of chloroform and 3 volumes of water and vigorous mixing. The denatured protein was removed by centrifugation at 15,000g for 1 min at 4 °C. The supernatant was dried in vacuo and dissolved in 20 μl water. Half of the extract was analysed by LC–MS. The tracer molecules were prepared by dephosphorylation of [15N]ATP and [15N]GTP as follows: 1,000 pmol each of [15N]ATP (Silantes) and [15N]GTP (Silantes) was treated with 0.04 U alkaline phosphatase (PAP, from Shewanella sp. SIB1, BioDynamics Laboratory) in 20 mM ammonium acetate (pH 8.0) at 60 °C for 30 min. After dephosphorylation, PAP was heat denatured at 95 °C for 5 min.

Construction of gene knockout strains of T. kodakarensis

Knockout strains of T. kodakarensis were constructed by pop-in/pop-out recombination as described previously64. The 5′ and 3′ flanking regions (about 1,000 bp) of T. kodakarensis arkI and kptA were PCR amplified from genomic DNA with a set of primers (Supplementary Table 6) and inserted into the pUD3 vector bearing the pyrF marker65 to yield pUD3-arkI and pUD3-kptA. The T. kodakarensis KU216 strain (ΔpyrF) was transformed with pUD3-arkI or pUD3-kptA, and the uracil-prototrophic transformants generated by pop-in recombination were selected on an ASW-AA-S0 plate without uracil. The selected strains were then cultured on an ASW-AA-S0 plate supplemented with 5-FOA to obtain uracil-auxotrophic, 5-FOA-resistant transformants formed by pop-out recombination. The knockout strains of arkI or kptA were selected among the transformants by genomic PCR with a set of primers (Supplementary Table 6). The double-knockout strain of arkI and queEarkI/queE::Tn) was constructed by deletion of arkI from FFH05 (queE::Tn) isolated from a random mutagenesis library19. T. kodakarensis strains used in this study are listed in Supplementary Table 7.

Growth phenotype analysis

T. kodakarensis KU216 (wild type), FFH05 (queE::Tn), ΔarkI and ΔarkI/queE::Tn strains were precultured in MA-YT-Pyr medium at 83 °C overnight and inoculated into 8 ml fresh MA-YT-Pyr medium with an initial OD600 of 0.01. The cells were cultured at 83 °C, 87 °C or 91 °C, and cell growth was monitored every 2 h by measuring OD600 with an S1200 diode array spectrophotometer. Graphs were generated using Microsoft Excel.

In vitro transcription of tRNA

For in vitro transcription of T. kodakarensis tRNAVal3 and its G5–C68 variants by T7 RNA polymerase66, template DNAs were constructed by PCR using synthetic DNA (Supplementary Table 6). The tRNAs were transcribed at 37 °C overnight in a reaction mixture consisting of 40 mM Tris-HCl (pH 7.5), 24 mM MgCl2, 5 mM DTT, 2.5 mM spermidine, 0.01% (vol/vol) Triton X-100, 0.8 μg ml–1 T7 RNA polymerase, 1 μg ml–1 pyrophosphatase, 30 nM DNA template, 2 mM ATP, 2 mM CTP, 2 mM UTP, 2 mM GTP and 10 mM GMP, followed by extraction with phenol/chloroform treatment and desalting with PD-10 columns (GE Healthcare). In vitro transcripts prepared in this way were separated by 10% denaturing PAGE, followed by staining with toluidine blue. The stained bands were cut out and eluted from the gel slice with elution buffer, followed by filtration to remove the gel pieces and ethanol precipitation.

In vitro phosphorylation of tRNA by ArkI

Up47 formation by TkArkI was carried out at 70 °C for 20 min in a reaction mixture (30 μl) containing 50 mM HEPES-KOH (pH 7.5), 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 10% (vol/vol) glycerol, 0.5 mM ATP, 0.9 μM tRNA fraction (from the T. kodakarensis ΔarkI strain) and 1 μM TkArkI. After the reaction, the tRNA was extracted by acidic phenol/chloroform, desalted on a NAP-5 column (GE Healthcare) and precipitated with isopropanol. For RNA-MS, the prepared tRNA was dialysed against water on a nitrocellulose membrane (0.025-μm VSWP, MF-Millipore, Merck) for 2 h (drop dialysis). To examine GTP as a phosphate donor, 0.5 mM ATP or GTP was added to the reaction mixture and Up47 formation was performed with 0.5 μM TkArkI for 5 min, followed by RNA-MS analysis. The activities of TkArkI variants were measured by γ-phosphate transfer from [γ-32P]ATP to tRNA similarly to the kinetic studies of TkArkI (see below). tRNA phosphorylation was performed at 70 °C for 15 min in an 8-μl reaction mixture. For PAGE analysis, 4 μl of the reaction mixture was mixed with 4 μl of 2× loading solution, resolved by 10% denaturing PAGE and exposed to an imaging plate to visualize radiolabelled RNA with an FLA-9000 imaging analyser (Fujifilm). The gel image was analysed using Multi Gauge (Fujifilm). Bar graphs with independent plots were prepared with R (R Foundation). For phosphorylation of total RNA, the reaction was performed at 70 °C for 30 min in an 8-μl reaction mixture consisting of 50 mM HEPES-NaOH (pH 7.5), 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 10% (vol/vol) glycerol, 100 μM [γ-32P]ATP (3,000 mCi mmol–1; PerkinElmer), 1.8 μM TkArkI and 50 ng μl–1 total RNA fraction (from the T. kodakarensis ΔarkI strain). Then, 0.5 μl of 50 mM EDTA (pH 8.0) was added, and 4 μl of reaction mixture was mixed with 2× loading solution, resolved by 10% denaturing PAGE and visualized as described above.

Formation of Up47 by other ArkI homologues was carried out at 70 °C for 30 min in a reaction mixture (30 μl) containing 50 mM PIPES-NaOH (pH 6.9), 125 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 10% (vol/vol) glycerol, 500 μM ATP, 0.05 mg ml–1 BSA (Takara), 1 μM tRNA transcript and 0.5 μM ArkI protein. For NvArkI, the reaction temperature was set to 45 °C. For ArkI homologue from N. profundicola (NpArkI), the reaction was carried out at 50 °C for 60 min. After the reaction, tRNA was prepared as described above. For PAGE analysis, Up47 formation was carried out in a reaction mixture (8 μl) containing 50 mM PIPES-NaOH (pH 6.9), 125 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 10% (vol/vol) glycerol, 100 μM [γ-32P]ATP (3,000 mCi mmol–1; PerkinElmer), 0.1 mg ml–1 BSA (Takara), 0.75 μM recombinant ArkI homologue (NpArkI, NvArkI or LeArkI) and 50 ng μl–1 E. coli total RNA. Then, the reaction mixture was mixed with 2× loading solution, resolved by 10% denaturing PAGE and visualized as described above.

In vitro dephosphorylation of tRNA by T. kodakarensis KptA

Dephosphorylation of Up47 by TkKptA was carried out at 60 °C for 1 h in a reaction mixture (30 μl) containing 20 mM Tris-HCl (pH 7.4), 0.5 mM EDTA (pH 8.0), 1 mM NAD+, 2.5 mM spermidine, 0.1 mM DTT, 0.9 μM T. kodakarensis tRNA fraction and 0.1 μg μl–1 recombinant TkKptA. After the reaction, the tRNA was extracted by acidic phenol/chloroform, desalted on a NAP-5 column (GE Healthcare) and precipitated with isopropanol. For RNA-MS, the prepared tRNA was desalted by drop dialysis as described above.

Kinetic studies of T. kodakarensis ArkI and KptA

TkArkI-mediated Up47 formation was quantified by γ-phosphate transfer from [γ-32P]ATP to tRNA. For kinetic measurement of the tRNA substrate, tRNA phosphorylation was performed at 70 °C in a reaction mixture (25 μl) consisting of 50 mM PIPES-NaOH (pH 6.9), 125 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 10% (vol/vol) glycerol, 100 μM [γ-32P]ATP (1,500 mCi mmol–1; PerkinElmer), 0.05 mg ml–1 BSA (Takara), 0.05 μM TkArkI and 0.1–5.0 μM of in vitro-transcribed T. kodakarensis tRNAVal3. For kinetic measurement of the ATP substrate, the ATP concentration was altered from 15.6 to 1,000 μM [γ-32P]ATP (750 mCi mmol–1; PerkinElmer) and the tRNA concentration was increased to 1.0 μM. At each time point (2 and 5 min), 8-μl aliquots were taken and mixed with an equal volume of 2× loading solution (7 M urea, 0.2% (wt/vol) bromophenol blue, 0.2% (wt/vol) xylene cyanol and 50 mM EDTA (pH 8.0)) to quench the reaction. Each sample was subjected to 10% denaturing PAGE. The gel was exposed on an imaging plate to measure radiolabelled tRNAs using an FLA-9000 imaging analyser. Kinetic parameters were calculated using Prism 7 (GraphPad).

TkKptA-mediated dephosphorylation of Up47 was quantified by measuring the reduction in radioactivity for tRNA. In vitro-transcribed T. kodakarensis tRNAVal3 was phosphorylated by TkArkI with [γ-32P]ATP as described above and then purified by gel extraction and isopropanol precipitation. In addition, the same tRNA was phosphorylated by TkArkI with unlabelled ATP. By mixing labelled and unlabelled tRNAs, the specific activity of the labelled tRNA was adjusted to 6,250 c.p.m. per pmol in buffer consisting of 50 mM HEPES-KOH (pH 7.6), 5 mM MgCl2 and 1 mM DTT. The labelled tRNA was incubated at 80 °C for 5 min and then cooled at room temperature, followed by isopropanol precipitation. The labelled tRNA was dissolved in water to a concentration of 8 μM (50,000 c.p.m. per μl). Dephosphorylation of the labelled tRNA by TkKptA was performed at 70 °C in a reaction mixture (30 μl) consisting of 50 mM PIPES-NaOH (pH 6.9), 125 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 10% (vol/vol) glycerol, 1 mM NAD+, 0.05 mg ml–1 BSA (Takara), 1 nM TkKptA and 12.5–800 nM 32P-labelled tRNA. At each time point (2 and 5 min), 8-μl aliquots were spotted on Whatman 3MM filter paper, which was immediately soaked in 5% (wt/vol) trichloroacetic acid. The filter paper was washed three times for 15 min with ice-cold 5% (wt/vol) trichloroacetic acid, rinsed for 5 min with ice-cold ethanol and dried in air. Radioactivity on the filter paper was measured by liquid scintillation counting (Tri-Carb 2910TR, PerkinElmer). Kinetic parameters were calculated using Prism 7.

In vivo dephosphorylation of Up47 by KptA

N. viennensis arkI was PCR amplified and cloned into pMW118 (Invitrogen) under the control of the synthetic constitutive J23106 promoter67,68, followed by insertion of sequences encoding a His6 tag and a 3×Flag tag at the C terminus of the N. viennensis arkI gene, yielding pMW-J23106-nvarkI (Supplementary Table 7). T. kodakarensis kptA, E. coli kptA and S. cerevisiae tpt1 were PCR amplified and cloned into pQE-80L (Qiagen). The ampicillin resistance cassette (Ampr) was replaced with a chloramphenicol resistance cassette (Camr), yielding pQE-80LC-tkkptA, pQE-80LC-eckptA and pQE-80LC-sctpt1, respectively (Supplementary Table 7). The E. coli ΔtrmBΔtapT (Kanr) strain was transformed with pMW-J23106-nvarkI and further transformed with pQE-80LC-tkkptA, pQE-80LC-eckptA or pQE-80LC-sctpt1. The transformants were inoculated in 3 ml LB supplemented with 20 μg ml–1 chloramphenicol, 50 μg ml–1 kanamycin and 100 μg ml–1 ampicillin and cultured at 37 °C until mid-log phase. When the OD610 reached 0.6, IPTG was added to a final concentration of 10 or 100 μM to induce expression of the KptA/Tpt1p homologue and cells were cultured for 3.5 h. A 1.5-ml aliquot of the culture was taken, and the tRNA fraction was extracted and analysed by shotgun RNA-MS as described above. Primers, E. coli strains and plasmids used are listed in Supplementary Tables 6, 7. Bar graphs with independent plots were prepared with R (R Foundation).

Drawing of chemical structures

Chemical structures were drawn with chemical structure drawing tools, including ACD/ChemSketch (ACD/Labs) or ChemDraw (PerkinElmer).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

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