Strains and medium

The organisms used in this study are listed in Supplementary Table 3. Antibiotics were used in the following concentrations: 200 μg ml−1 streptomycin, 50 μg ml−1 carbenicillin, 50 μg ml−1 kanamycin (Km) and 1 μg ml−1 chloramphenicol (Cm).

E. coli strain BW25113 was cultured in LB medium at 37 °C for 18 h. Bacillus subtilis strain 168 was cultured in LB medium at 37 °C for 24 h. P. putida NITE Biological Resource Center (NBRC) 14164, Geobacillus kaustophilus NBRC 102445 and Acidimicrobium ferrooxidans DSM 10331 were obtained from NBRC. P. putida was cultured in NBRC 702 medium (10 g l−1 tryptone, 2 g l−1 yeast extract and 1 g l−1 MgSO4·7H2O) at 30 °C for 22.5 h. G. kaustophilus was cultured in NBRC 702 medium at 55 °C for 23 h. A. ferrooxidans was cultured in 0.5 g l−1 MgSO4·7H2O, 0.4 g l−1 (NH4)2SO4, 0.2 g l−1 K2HPO4, 0.1 g l−1 KCl, 10 mg l−1 FeSO4·7H2O and 0.25 g l−1 yeast extract (adjusted to pH 2.0 with 2 M H2SO4) at 45 °C. Mycoplasma mobile, kindly provided by M. Miyata (Osaka City University), was grown in Aluotto medium (pH 7.8), which was composed of 2.1% heart infusion broth (Difco), 0.56% yeast extract, 10% horse serum (inactivated at 56 °C) and 0.005% ampicillin at 25 °C. Cell pellets of Haloarcula marismortui were kindly provided by T. Fujiwara (Shizuoka University). V. cholerae and V. parahaemolyticus were cultured in LB or M9 medium at 37 °C. A. hydrophila was cultured in nutrient broth (Difco) at 30 °C overnight. S. oneidensis MR-1 was cultured in Tryptic soy broth (Difco) at 30 °C overnight.

Saccharomyces cerevisiae BY4742 (Euroscarf) was cultured in 1% yeast extract, 2% peptone and 2% glucose at 30 °C for 18 h. Spinach (S. oleracea) was purchased from a grocery store. A. thaliana Col-0 was purchased from Inplanta Innovations. Tobacco BY-2 cells (RIKEN BRC through the National BioResource Project) were cultured in modified Linsmaier and Skoog medium (pH 5.5), which contained Murashige and Skoog Plant Salt Mixture (Wako), 30 g l−1 sucrose, 0.2 g l−1 KH2PO4, 100 mg l−1 myo-inositol, 1 mg l−1 thiamine-HCl, 0.2 mg l−1 2,4-dichlorophenoxyacetic acid and NaOH, in the dark at 25 °C with agitation at 130 rpm. The unicellular red alga C. merolae 10D was cultured in MA2 medium (pH 3)47,48 at 42 °C under LED light.

Construction of plasmids and strains

The V. cholerae Para-tilS strain was created using homologous recombination and a derivative of the suicide vector pCVD442. The arabinose-inducible promoter with the araC gene (pBAD; Invitrogen) and ~1,000 base pairs of DNA flanking each side of the target were cloned into the SmaI site of pCVD442 using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs (NEB)). The endogenous tilS promoter was replaced with the arabinose-inducible promoter with araC.

E. coli and V. cholerae TilS protein-expressing vectors (pET28b-ECTilS and pET28b-VCTilS) were generated by integrating the E. coli or V. cholerae TilS open reading frame into linearized pET28 using NEBuilder HiFi DNA Assembly Master Mix (NEB).

Reporter protein-expressing plasmids for measuring Ile-decoding ability were generated by integrating tandem sequences testing decoding of the Ile codon into linearized pMMB207 encoding mCherry and bright GFP using NEBuilder HiFi DNA Assembly Master Mix (NEB). The DNA primers used to construct plasmids and strains are listed in Supplementary Table 4.

RNA extraction

For A. ferrooxidans, budding yeast, S. pombe, A. thaliana and spinach cells were frozen with liquid nitrogen followed by homogenization with a prechilled mortar and pestle. The cell powder was suspended in a 1:1 mixture of water–saturated phenol and extraction buffer (50 mM NaOAc and 10 mM Mg(OAc)2, pH 5.2) and vigorously stirred for 1 h at room temperature. SDS and sarkosyl were added as needed. For B. subtilis and cyanobacteria, cells suspended in a 1:1 mixture of water–saturated phenol and extraction buffer were incubated at 95 °C for 20 min. For E. coli, M. mobile, P. putida, G. kaustophilus, C. merolae, V. cholerae and H. marismortui cells suspended in a 1:1 mixture of water–saturated phenol and extraction buffer were subjected to two freeze-thaw cycles using liquid N2, followed by vigorous stirring for 1 h at room temperature. The aqueous phase was separated by centrifugation and washed with chloroform. RNA was precipitated with 2-propanol. RNA dissolved in ultrapure water was cleaned up with TriPure (Roche) and precipitated with ethanol. The pellet was rinsed with 80% ethanol and dried. The obtained RNA was further separated by anion-exchange chromatography with DEAE Sepharose Fast Flow (Cytiva) or by polyacrylamide gel electrophoresis and gel slicing to enrich tRNAs. The tRNA mixture of Thermus thermophilus HB27 was kindly provided by N. Shigi (AIST).

tRNA isolation

V. cholerae tRNAIle2 was isolated by a batch-wise solid-phase DNA probe method49 from the total RNA fraction separated by anion-exchange chromatography. Typically, 2 mg of the RNA fraction was mixed with 200–400 μl of streptavidin agarose beads (Pierce) bound to 4 nmol of the biotinylated DNA probe (Supplementary Table 5) in 300 mM HEPES–KOH (pH 7.0), 1.2 M NaCl, 15 mM EDTA and 1 mM DTT at 68 °C for 30 min with shaking. The beads were washed three times with 15 mM HEPES–KOH (pH 7.0), 0.6 M NaCl, 7.5 mM EDTA and 1 mM DTT and seven times with 0.5 mM HEPES–KOH (pH 7.0), 20 mM NaCl, 0.25 mM EDTA and 1 mM DTT. Purified tRNAs were extracted from the beads with TRIzol (Thermo Fisher Scientific). After treating with Turbo DNase (Thermo Fisher Scientific) to remove residual DNA probes, tRNA was purified by 10% PAGE with 7 M urea.

tRNA sequences for isolation were obtained from PlantRNA50, tRNADB-CE51, tRNAdb52 and NCBI, compared and integrated (Supplementary Data 1). Chloroplast and/or mitochondrial tRNAsIle2 from spinach, A. thaliana and C. merolae were homogeneously isolated by RCC using an automated RCC device, basically following the previously described protocol31,32. DNA probes complementary to each tRNA were designed using Raccess53 to have sufficient binding capability and specificity. The sequences of the DNA probes used in this study are listed in Supplementary Table 5. The 5′-EC amino-modified DNA probes (Sigma-Aldrich) were covalently immobilized on NHS-activated Sepharose 4 Fast Flow (Cytiva). The DNA resins packed in custom-made tips were set to a custom-made multichannel head on the RCC device. The tips were cleaned up with 50 mM NaOH before each RCC run. RNA dissolved in 6× NME buffer (1.2 M NaCl, 30 mM MES–NaOH (pH 6.0), 15 mM EDTA and 1 mM DTT) was passed through the tip by auto-pipetting at 65 °C. After washing the tip columns with 0.1× NME at 40 °C, bound tRNAs were eluted in 0.1× NME at 68 °C. Purity was confirmed by 10% PAGE with 7 M urea.

Nucleoside analysis by MS

Four micrograms of total tRNA or ~10 pmol of isolated tRNA were digested with 0.05 U nuclease P1 (FUJIFILM Wako Pure Chemical) and 0.04 U bacterial alkaline phosphatase (BAP; from E. coli C75; Nippon Gene) in 20 mM NH4OAc (pH 5.3) at 37 °C for 1 h.

For normal-phase chromatography, hydrophilic interaction LC (HILIC)/ESI–MS was used for nucleoside analysis54. Nucleosides were dissolved in 90% acetonitrile/10% water and applied to a ZIC-cHILIC column (3 μm particle size, 2.1 × 150 mm; Merck Millipore) coupled with ESI–MS on a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific), equipped with an ESI source and an Ultimate 3000 LC system (Thermo Fisher Scientific). The mobile phase consisted of 5 mM NH4OAc (pH 5.3; solvent A) and acetonitrile (solvent B). The nucleosides were chromatographed with a flow rate of 100 μl min−1 in a multistep gradient as follows: linear 90–85% solvent B from 0 to 10 min, 85–30% solvent B from 10 to 30 min with curve 7, 30% solvent B for 10 min and then initialized to 90% solvent B. Proton adducts of nucleosides were scanned in a positive polarity mode over an m/z range of 103–700 or 900. Xcalibur 4.4 (Thermo Fisher Scientific) was used for system operation.

For reverse-phase chromatography/ESI–MS55, nucleosides were applied to a SunShell C18 column (2.6 μm particle size, 2.1 × 150 mm; ChromaNik Technologies) and analyzed by a Q Exactive system with the same solvents as described above. The nucleosides were chromatographed with a flow rate of 75 μl min−1 in a multistep gradient as follows: 0–15% solvent B from 0 to 30 min with curve 7, linear 15–60% solvent B from 30 to 35 min, 60% solvent B for 10 min and then initialized to 0% solvent B.

For Figs. 1f and 2a,b and Extended Data Figs. 5 and 6a, nucleosides of tRNAs or tRNA fractions were analyzed by dynamic multiple reaction monitoring (MRM) using Agilent 6460 QQQ (Agilent). One hundred nanograms of tRNA fraction or purified tRNA were digested with 0.5 U nuclease P1 (US Biological) and 0.1 U phosphodiesterase I (Sigma) in 22 μl reactions containing 50 mM Tris–HCl (pH 5.3) and 10 mM ZnCl2 at 37 °C for 1 h. Reaction mixtures were then mixed with 2 μl of 1 M Tris–HCl (pH 8.3) and 1 U μl−1 calf intestine phosphatase (Sigma) and incubated at 37 °C for 30 min. Enzymes were filtered out using 10K ultrafiltration columns (VWR). Then, 18 μl aliquots were mixed with 2 μl of 50 μM 15N-dA, and 2.5–10 μl digests were injected into an Agilent 1290 ultra-HPLC system bearing a Synergi Fusion-RP column (100 × 2 mm, 2.5 μm; Phenomenex) at 35 °C with a flow rate of 0.35 ml min−1 using a solvent system consisting of 5 mM NH4OAc (buffer A) and 100% acetonitrile (buffer B). The gradient of acetonitrile was as follows: 0%, 0–1 min; 0–10%, 1–10 min; 10–40%, 10–14 min; 40–80%, 14–15 min; 80–100%, 15–15.1 min; 100%, 15.1–18 min; 100–0%, 18–20 min and 0%, 20–26 min. The eluent was ionized by an ESI source and directly injected into the mass spectrometer with the following parameters: gas temperature, 250 °C; gas flow, 11 l min−1; nebulizer, 20 psi; sheath gas temperature, 300 °C; sheath gas flow, 12 l min−1; capillary voltage, 1,800 V and nozzle voltage, 2,000 V.

Dynamic MRM was carried out to survey known RNA modifications. The retention time windows and m/z values of precursor and product ions for dynamic MRM analyses are listed in Supplementary Data 2.

RNA fragment analysis by MS

For RNA fragment analysis, 1 pmol of isolated tRNA was digested with 20 U RNase T1 (Thermo Fisher Scientific) in 20 mM NH4OAc (pH 5.3) at 37 °C for 1 h. The digests were mixed with a one-tenth volume of 0.1 M triethylamine acetate (pH 7.0) and subjected to LC-nano ESI–MS on an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a splitless nanoflow HPLC (nano-HPLC) system (DiNa; KYA Technologies) using a nano-LC trap column (C18, 0.1 × 0.5 mm; KYA Technologies) and a capillary column (HiQ Sil C18W-3, 0.1 × 100 mm; KYA Technologies)32,55. Digested fragments were separated for 35 min at a flow rate of 300 nl min−1 by capillary LC using a linear gradient from 2% to 100% solvent B in a solvent system consisting of 0.4 M 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; pH 7.0; solvent A) and 0.4 M HFIP (pH 7.0) in 50% methanol (solvent B). The eluent was ionized by an ESI source in a negative polarity mode and scanned over an m/z range of 600–2,000. Xcalibur 2.0.7 (Thermo Fisher Scientific) was used for system operation. The LC–MS data were analyzed using Qual Browser (Thermo Fisher Scientific). Excel was used to calculate the m/z value of each fragment.

Purification of N341 nucleoside from spinach

Forty kilograms of fresh spinach were freeze-dried without blanching by Miyasaka Brewing Company and powdered by Mikasa Sangyo. Total RNA was extracted from 1.5 kg spinach powder with 15 l extraction buffer (50 mM NaOAc, 10 mM Mg(OAc)2, 0.2% SDS, 0.2% sarkosyl and 28.8 mM 2-mercaptoethanol (pH 5.2)) and 15 l water–saturated phenol by stirring with a mechanical stirrer for 3 h at room temperature. After centrifugation, the recovered upper phase was extracted using chloroform and subjected to 2-propanol precipitation. The RNA pellet was dissolved in ddH2O and purified by the AGPC method56. RNA was precipitated with 2-propanol, rinsed with 70% ethanol and dried. The obtained total RNA (total 14.4 g) was applied to a DEAE Sepharose FF column (Cytiva) equilibrated with buffer A (10 mM HEPES–KOH (pH 7.5) and 250 mM NaCl), washed with buffer A and then eluted with buffer B (10 mM HEPES–KOH (pH 7.5) and 1 M NaCl) to remove contaminants and long RNAs57.

Spinach chloroplast tRNAIle2 was isolated from the obtained RNA by chaplet column chromatography33. In total, 400 nmol of 3′-EC-amino linker DNA probe (Supplementary Table 5) was immobilized on HiTrap NHS-activated HP columns (1 ml; Cytiva). In total, 3.26 mg tRNAIle2 was isolated.

The isolated tRNAIle2 was digested in 20 mM NH4OAc (pH 5.3) containing nuclease P1 (0.1 U per 40 μg tRNA) and BAP (0.15 U per 40 μg tRNA) at 37 °C. The solution was purified with a PoraPak Rxn RP column (Waters) to remove salts, enzymes and other pyrimidines. The column was washed with 5 mM NH4OAc (pH 5.3) buffer and eluted with 50% CH3CN. The eluates were dried and separated by reverse-phase HPLC with an HP1100 LC system (Agilent Technologies) equipped with an Inertsil ODS-3 column (5 μm, 10 mm × 250 mm; GL Sciences). The mobile phase consisted of 5 mM NH4OAc (pH 7.2; solvent A) and 60% acetonitrile (solvent B). The nucleosides were chromatographed with a flow rate of 1 ml min−1 in a multistep linear gradient as follows: 0–14% solvent B from 0 to 2 min, 14% solvent B for 15 min, 14–21% solvent B from 17 to 45 min, 21–99% solvent B from 45 to 55 min and then 99% solvent B for 20 min. The N341-rich fraction that was eluted around 50 min was collected (Supplementary Fig. 3a), dried and dissolved in 80% acetonitrile. The fraction was further purified using a ZIC HILIC column (5 μm, 10 mm × 150 mm; Merck Millipore) with a mobile phase of 5 mM NH4OAc (pH 5.3; solvent A) and acetonitrile (solvent B). N341 was separated with a flow rate of 0.2 ml min−1 in a multistep linear gradient as follows: 85–30% solvent B from 0 to 40 min, 30% solvent B for 10 min and then initialized to 85% B. N341 that eluted around 36 min was collected (Supplementary Fig. 3b), dried and desalted with a PoraPak Rxn RP column. Purity was confirmed by LC–MS analysis. The yield of N341 was about 0.16 A260 units.

Deuterium exchange MS

One nanomole of purified N341 was dissolved in D2O (D, 99.9%; Cambridge Isotope Laboratories), incubated at 40 °C for 15 min and dried under a vacuum. This operation was repeated twice more. Deuterium-substituted N341 was dissolved in 50% acetonitrile/D2O (D, 99.96%; Cambridge Isotope Laboratories) and directly infused into an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific).

Chemical synthesis of ava2C and NMR spectroscopy

The ava2C nucleoside was chemically synthesized by the scheme shown in Fig. 2f. 2-Thiocytidine was prepared as previously reported58. The other materials were purchased from commercial sources. 2-Thiocytidine (15.4 mg, 0.06 mmol) was mixed with sodium bicarbonate (4.4 mg, 0.052 mmol; FUJIFILM Wako Pure Chemical) and iodomethane (6.5 μl, 0.104 mmol; TCI) in 0.5 ml dried ethanol. The solution was incubated for 1 day to obtain 2-methylthiocytidine and centrifuged. The supernatant was collected and concentrated. To the residue, 5-AVA hydrochloride (7.9 mg, 0.052 mmol; Enamine) and sodium hydroxide (4.2 mg, 0.104 mmol; FUJIFILM Wako Pure Chemical) were added and then incubated in 1 ml dried ethanol for 3 days. Water and acetic acid were added to the final solution to adjust the pH and volume to 7 and 5 ml, respectively. The product was fractionated and purified by reverse-phase chromatography with an ODS column (COSMOSIL 5C18-MS-II; Nacalai Tesque; Extended Data Fig. 7a) using a gradient of triethylamine acetate buffer (0.2 M (pH 7.0); solvent A) to acetonitrile (0–10% solvent B from 0 to 20 min, 10–30% solvent B from 20 to 25 min and 30% solvent B from 25 to 30 min) to give 6.58 mg ava2C as a white powder (32% yield) after lyophilization. The product was analyzed by LC–MS (Fig. 2g and Supplementary Fig. 5) and NMR. 1H and COSY NMR spectra were measured with a JEOL JNM-ECS 400 instrument (Extended Data Fig. 7c–e). The chemical shifts are shown in parts per million using tetramethylsilane or solvent (DMSO-d6) as an internal standard. For 1H NMR (400 MHz, DMSO-d6; Extended Data Fig. 7c), the shifts are as follows: δ = 1.43–1.66, 2.07, 3.36 (11H, CH2 in AVA, 2′-OH, 3′-OH, 5′-OH), 3.54–3.75 (m, 2H, H5′), 3.92–4.13 (m, 3H, H4′, H3′, H2′), 5.71 (d, J = 5.3 Hz, 1H, H1′), 6.02 (d, J = 7.6 Hz, 1H, H5), 6.72 (s, 1H, 4-NH), 7.34 (s, 1H, 2-NH) and 8.02–8.32 (m, 3H, H6, CONH2 in AVA). For 1H NMR (400 MHz, DMSO-d6 + D2O; Extended Data Fig. 7d), the shifts are as follows: δ = 1.44–1.67, 2.11, 3.40 (8H, CH2 in AVA), 4.07 (ddd, J = 12.5, 5.2, 2.6 Hz, 2H, H4′, H3′), 4.18–4.24 (m, 1H, H2′), 5.62 (d, J = 6.3 Hz, 1H, H1′), 6.17 (d, J = 7.6 Hz, 1H, H5) and 8.07 (d, J = 7.7 Hz, 1H, H6). No exchangeable protons of amine (typically δ = 0.5–5) were observed. An exchangeable proton estimated to be at position N4 was observed. These findings indicate that the amino group of 5-AVA is bound to the C2 atom of the cytosine ring. LC–MS analysis, the calculated mass for the (M + H)⁺ ion of ava2C was 342.1777 (C14H23N5O5), and the observed mass was 342.1781.

UV spectra of ava2C and L

The synthetic ava2C nucleoside was dissolved in 50 mM sodium phosphate buffer (pH 2, 3 and 6–9), sodium acetate (pH 4 and 5) or sodium borate (pH 10). UV spectra were measured with a BioDrop DUO+ instrument (Biochrom). The UV spectrum of L (NARD Institute) was also measured.

Enzymatic reconstitution of ava2C, L, and t6A

The E. coli tRNAIle2 transcript was synthesized by T7 run-off transcription59,60. Reconstitution of ava2C or L was carried out at 37 °C for 1 h in a reaction mixture containing 100 mM HEPES–KOH (pH 8.6), 50 mM KCl, 2 mM ATP, 2 mM DTT, 10 mM 5-AVA hydrochloride or l-lysine monohydrochloride (FUJIFILM Wako Pure Chemical), 1 μg transcribed tRNA and 1.5 μM E. coli TilS60. tRNAs were extracted with TriPure (Roche), precipitated twice with ethanol and rinsed twice with 80% ethanol.

Reconstitution of the t6A modification was carried out at 37 °C for 1.5 h in a reaction mixture containing 100 mM HEPES–KOH (pH 7.6), 25 mM MgCl2, 25 mM KCl, 5 mM DTT, 2 mM ATP, 10 mM NaHCO3, 5 mM l-threonine, 2.5 μM transcribed tRNA and 2.5 mM each E. coli TsaC, TsaD, TsaE and TsaB60. After the reaction, the tRNA was purified using a mixture of acidic phenol–chloroform–isoamyl alcohol (25:24:1), followed by NAP-5 gel filtration (Cytiva) and ethanol precipitation. The frequency of each modification introduced was monitored by LC–MS analysis.

For Supplementary Fig. 6, the in vitro reaction was conducted with V. cholerae tRNAIle2 transcript, E. coli and V. cholerae TilS and a small compound fraction derived from V. cholerae cells. In a 20 μl reaction, 50 pmol tRNAIle2 transcript was mixed with 1 μM E. coli or V. cholerae TilS, 1 mM ATP, 10 mM MgCl2 and 7 μl small compound fraction and incubated at 37 °C for 1 h. Reacted tRNAs were extracted using TRIzol (Thermo Fisher Scientific) and analyzed by LC–MS as described above.

Aminoacylation assay

Isoleucylation of each tRNA was carried out at 37 °C in a reaction mixture consisting of 100 mM Tris–HCl (pH 7.8), 5 mM MgCl2, 10 mM KCl, 1 mM DTT, 2 mM ATP, 50 μM L-(U-14C) Ile (12.025 GBq mmol−1; Moravek Biochemicals), 0.4 μM tRNA and 1.68 μM recombinant E. coli IleRS. At different time points, an aliquot was spotted onto a Whatman 3MM filter, and radioactivity was measured by a liquid scintillation counter (PerkinElmer) as previously described60.

Small compound fraction from V. cholerae cells

Five milliliters of a V. cholerae overnight culture were inoculated into 1 l LB medium and cultured at 37 °C until optical density (OD)600 reached 0.4. Cells were harvested by centrifugation and resuspended in 15 ml lysis buffer (300 mM NaCl, 10% glycerol, 50 mM Tris–HCl (pH 8.1) and 10 mM MgCl2) containing 6 U DNase I. Cells were disrupted using an Emulsiflex instrument (Avestin) and cleared by centrifugation. Then, 500 μl lysate was loaded onto a YM-10 Amicon filter (Merck Millipore) and spun at 4,000g for 30 min. The flowthrough fraction was collected and stored at −80 °C.

Recombinant proteins

For E. coli and V. cholerae TilS, the BL21(DE3) strain transformed with pET28b encoding E. coli tilS or V. cholerae tilS was grown in 10 ml LB medium (50 μg ml−1 Km) overnight, inoculated into 1 l LB medium (50 μg ml−1 Km) and grown at 37 °C with shaking. When OD600 reached 0.3, the flask was chilled to 18 °C and shaken for 30 min. Protein expression was induced by the addition of 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and the flask was incubated with shaking at 18 °C for 24 h. Harvested cells were resuspended in 40 ml lysis buffer (50 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 10% glycerol, 300 mM NaCl, 0.2 U ml−1 DNase I, 1 mM phenylmethylsulfonyl fluoride and complete proteinase inhibitor mixture (Roche)) and homogenized with an EmulsiFlex instrument (Avestin) for 20 min. The cleared lysate (35 ml) supplemented with 700 µl of 2 M imidazole (final concentration 40 mM) was mixed with 1.5 ml Ni-NTA beads equilibrated with 10 ml lysis buffer and incubated at 4 °C for 2.5 h with gentle rotation. Protein-bound beads were loaded on an open column (Bio-Rad) and washed twice with 10 m wash buffer (50 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 10% glycerol, 300 mM NaCl and 40 mM imidazole). Protein was eluted with elution buffer 1 (50 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 10% glycerol, 300 mM NaCl and 250 mM imidazole) and elution buffer 2 (50 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 10% glycerol, 300 mM NaCl and 400 mM imidazole). The two elution fractions were mixed and dialyzed overnight in dialysis buffer 1 (20 mM Tris–HCl (pH 8.0), 300 mM NaCl, 10% glycerol and 1 mM DTT) and 8 h in dialysis buffer 2 (20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10% glycerol and 1 mM DTT). The protein concentration was measured by Qubit (Invitrogen).

Reporter assay

The V. cholerae Para-tilS strain was transformed with the mCherry-GFP reporters harboring tandem Ile codons. Cells were cultured overnight in 2 ml LB medium (1 μg ml−1 Cm) at 30 °C and then diluted to OD600 = 0.01 in 5 ml LB medium containing 100 µM IPTG and 0.2% or 0.02% arabinose. Cells were cultured at 37 °C and harvested by centrifugation when OD600 reached 0.4. Harvested cells were resuspended in 100 μl PBS, mixed with 33 μl of 16% paraformaldehyde and incubated for 20 min at room temperature for fixation. Fixed cells were spun down, resuspended in 1 ml of PBS and left at room temperature overnight. The cell suspension was diluted 100-fold in 1 ml PBS and analyzed with a fluorescence-activated cell sorter (Sony, SH800S). GFP and mCherry signals were measured in 100,000 particles and analyzed with a custom R script. To decrease the background, particles with a signal intensity of less than 500 in the GFP or mCherry channel were excluded from the analysis. Relative log2(GFP/mCherry) values were used to evaluate the decoding activity of Ile codons.

Metabolic labeling

V. cholerae cells were cultured in 500 μl of LB medium at 30 °C overnight and washed with 500 μl of M9 medium twice. Then, 5 μl of inoculum was mixed with M9 medium or M9 medium supplemented with full-label lysine (13C6, 15N2-lysine) or mono-labeled lysine (1-13C-lysine) and then cultured at 37 °C with shaking for 22 h. The tRNA fraction was enriched by removing long RNAs by precipitation with a low concentration of isopropanol61. Briefly, 250 μl of total RNA in 300 mM NaOAc (pH 5.5) was mixed with 200 μl of isopropanol, incubated at room temperature for 10 min and then centrifuged at 20,400g for 10 min at room temperature. The supernatant was collected, mixed with 50 μl of isopropanol and incubated at −20 °C for 30 min. A small RNA fraction was recovered by centrifugation. The enriched small RNA fraction was digested into nucleosides and analyzed by LC–MS as described above.

Grid preparation and cryo-EM data collection

70S ribosomes were purified from E. coli MRE600 as previously described27. A series of synthetic mRNAs with or without 2′-OH substitutions at the fourth residue were purchased from Ajinomoto Bio-Pharma Service. The mRNA sequences are given in Supplementary Table 4. All mRNAs were gel purified by 10% PAGE with 7 M urea.

For complex formation, E. coli 70S ribosomes were mixed with mRNA and P-site tRNA in a solution containing 20 mM HEPES–KOH (pH 7.6), 10 mM Mg(OAc)2, 30 mM NH4Cl, 6 mM β-mercaptoethanol, 50 nM 70S ribosome, 500 nM mRNA and 500 nM P-site tRNA (E. coli tRNA-Glu or P. putida tRNAIle2) at 37 °C for 30 min. Then, 500 nM of P. putida tRNAIle2 was added and further incubated for 15 min. The resultant complexes were stabilized on ice for 30–60 min before grid preparation.

The grids were prepared using Vitrobot Mark IV (FEI) at 4 °C and 100% humidity. Quantifoil R1.2/1.3 300 mesh copper grids (Quantifoil) with a homemade thin carbon film were glow-discharged at 7 mA for 10 s with a PIB-10 Plasma Ion Bombarder (Vacuum Device). Thereafter, 3 µl of ribosome complex was applied to the grid, incubated for 30 s, blotted for 3 s with a blotting force of −10 and plunge-frozen in liquid ethane.

Automated data acquisition was performed using EPU 2.9 software (FEI) on a Krios G4 transmission electron microscope (FEI) operated at 300 kV. Images were acquired at the nominal magnification of ×105,000 with a defocus of 0.5–2.5 µm using a K3 direct electron detector (Gatan) in CDS-counting mode (0.8285 Å per pixel). The numbers of collected images for each ribosome complex are shown in Extended Data Fig. 9a. The collected images were fractionated into 48 frames with a total dose of 50 e− Å−2.

Image processing

Cryo-EM data processing was processed using RELION-3.1.2 (ref. 62). The movie frames were aligned with MotionCor2 (implemented with RELION), and CTF parameter estimation was performed with CTFFIND-4.1 (ref. 63). Particles were auto-picked using crYOLO64 with a box size of 530 pixels. The entire image processing procedure is summarized in Extended Data Fig. 9a.

For the ribosome complexed with A- and P-site P. putida tRNAIle2, 801,672 particles were extracted from 6,134 images in a box size of 150 pixels (2.9274 Å per pixel). Then, 2D classification was performed, and subsets of 70S ribosomes were selected. A refined 3D map was used as a consensus map and subjected to 3D classification. Particles classified as well-resolved 70S ribosomes with sufficient P-site density were kept, and a 3D-refined volume was generated to perform A-site-focused 3D classification using a mask. Particles in the subsets with high occupancy at the A-site were selected, re-extracted in a box size of 530 pixels (without rescaling, 0.8285 Å per pixel) and subjected to 3D refinement followed by per-particle CTF refinement, Bayesian polishing and second 3D refinement. The generated map was sharpened by postprocessing, and the final resolution was 2.25 Å. Image processing for ribosome complexes with different mRNAs with 2′-OH substitution at the residue 3′-adjacent to the A-site codon was conducted using the same workflow as described above. The final resolutions of these complexes ranged from 2.39 to 2.47 Å.

Model building

A starting model was assembled using published structures (Protein Data Bank (PDB) IDs: 7K00 (ref. 65) for 70S ribosome and 4V8N ref. 41 for A-site tRNAIle2 and mRNA) and docked into the final map by Chimera66, followed by real-space refinement by Phenix67. The model of P-site E. coli tRNAGlu was built by modifying the nucleotide sequence of tRNAfMet (PDB ID: 7K00) with Coot68. The ligand restraints for tRNA modifications were generated by eLBOW implemented with Phenix.

A-site binding assay

The A-site tRNA-binding assay was performed according to a previous study11,60 with modifications. For Fig. 3d, E. coli tRNAIle2 transcripts bearing t6A37 and ava2C34 or L34 (50 pmol) were dephosphorylated in a 10 µl reaction mixture containing 0.05 U BAP and 10 mM HEPES–KOH (pH 7.6) at 55 °C for 30 min and gel purified. tRNA was 3′-labeled with (γ-32P)ATP (PerkinElmer) by T4 polynucleotide kinase (Toyobo) according to the manufacturer’s instructions and gel purified. Radioactivity was quantified by Cherenkov counting. mRNA containing the AUA or AUG codon was synthesized by T7 run-off transcription59. The mRNA sequences are given in Supplementary Table 4. The P-site of the E. coli 70S ribosome was occupied with native E. coli tRNAGlu. A 10 µl mixture containing 2.5 pmol 70S ribosome, 20 pmol E. coli tRNAGlu and 25 pmol mRNA in binding buffer (50 mM HEPES–KOH (pH 7.6), 60 mM KCl, 6.5 mM Mg(OAc)2, 1 mM DTT and 0.5 mM spermine) was incubated at 37 °C for 30 min. Then, the tRNA solution (10 µl) containing 2.5 pmol 3′-32P-labeled tRNA (20,000 cpm) in binding buffer was added to the mixture, followed by further incubation at 37 °C for 15 min. The same mixture without mRNA was used as a negative control. The mixture (20 µl) was dot-blotted onto double-layered nitrocellulose (Protran Premium; Cytiva) and nylon (Hybond-N+; Cytiva) membranes and washed twice with 200 µl binding buffer. The membranes were exposed to an imaging plate, and radioactivity on the spots was visualized by a FLA-7000 image analyzer (Fujifilm) and quantified with Multi Gauge V3.0 (Fujifilm).

For Fig. 5b, a series of synthetic mRNAs with different 2′-OH substitutions at the residue 3′-adjacent to the A-site codon were purchased from Ajinomoto Bio-Pharma Service. The mRNA sequences are given in Supplementary Table 4. All mRNAs were gel purified by 10% PAGE with 7 M urea. P. putida tRNAIle2 was 3′-labeled with (γ-32P) ATP and T4 polynucleotide kinase (Toyobo). First, the P-site of the E. coli 70S ribosome was occupied with E. coli tRNAGlu at 37 °C for 30 min in a mixture (5 µl) containing 0.3 pmol E. coli 70S ribosome, 3 pmol E. coli tRNAGlu and 10 pmol mRNA in binding buffer (50 mM HEPES–KOH (pH 7.6), 120 mM KCl, 6.5 mM Mg(OAc)2, 1 mM DTT and 0.5 mM spermine). Then, the tRNA solution (5 µl) containing 0.5 pmol 3′-32P-labeled tRNA (5,000 cpm) in binding buffer was added to the mixture, followed by further incubation at 37 °C for 30 min. No mRNA was used as a negative control. The mixture (10 µl) was added to 60 µl binding buffer, dot-blotted onto double-layered nitrocellulose (GE Healthcare) and nylon (Hybond-N+; GE Healthcare) membranes and then washed three times with 200 µl binding buffer. The membranes were exposed to an imaging plate, and radioactivity on the spots was visualized by a FLA-7000 image analyzer (Fujifilm) and quantified with Multi Gauge V3.0 (Fujifilm). The binding ratio was calculated, and the statistical test was performed using Microsoft Excel.

Figure preparation

All figures were prepared with Canvas X (Nihon Poladigital K.K.) using the outputs of other softwares. Chemical structures were drawn with ChemDraw (PerkinElmer). The density maps and atomic models in Figs. 4 and 5 and Extended Data Figs. 9 and 10 were generated with Chimera and ChimeraX69. Bar graphs were generated using GraphPad Prism 7.04, 8 and 9.3.1 (GraphPad Software).

Reporting summary

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