Multiplex suppression of four quadruplet codons via tRNA directed evolution

General methods

Antibiotics (Gold Biotechnology) were used at the following working concentrations unless otherwise noted: carbenicillin, 50 μg/mL; spectinomycin, 100 μg/mL; chloramphenicol, 40 μg/mL; kanamycin, 30 μg/mL; tetracycline, 10 μg/mL; streptomycin, 50 μg/mL. David Rich Medium (DRM)²⁰ was used for PACE and all experiments involving plate reader measurements, excluding experiments involving JX33 RF1 knockout strain. For all other purposes, including phage-based selection assays, general cloning, and all experiments involving JX33 RF1 knockout strain, 2xYT media was used. All PCRs were performed using Phusion U HotStart DNA Polymerase (Life Technologies). Key plasmids from this study have been deposited on Addgene. See the Extended Supplement for all plasmids and plasmid maps, Addgene links, catalog numbers of materials, and plasmids used to produce each figure.

Chemically competent cell preparation

Strain S3489, a K12 derivative of S2060⁸¹ further optimized for directed evolution by deletion of ribosome hibernation-promoting factor Hpf, was used in all reporter assays, phage propagation assays, plaque assays, and PACE campaigns unless otherwise noted. To prepare competent cells, an overnight culture was diluted 1,000-fold into 50 mL of 2xYT media supplemented with maintenance antibiotics and grown at 37 °C with shaking at 230 × g to OD₆₀₀ ~0.4–0.6. Cells were pelleted by centrifugation at 6,000× RCF for 10 min at 4 °C. The cell pellet was then resuspended by gentle stirring in 5 mL of TSS (LB media supplemented with 5% v/v DMSO, 10% w/v PEG 3350, and 20 mM MgCl₂). The cell suspension was stirred to mix completely, aliquoted, and flash-frozen in liquid nitrogen, and stored at −80 °C until use.

USER cloning

Plasmids were cloned using USER (Uracil-Specific Excision Reagent) assembly, wherein primers were designed to include a USER junction, denoting the region between the 5′ primer end containing a dA and a deoxyuracil base approximately 15 base pairs downstream. USER junctions were additionally designed to have a 55 °C < T_m < 60 °C and minimal secondary structures. PCR products were run on a 1% agarose gel containing approximately 0.2 µg/mL ethidium bromide, allowing visualization under ultraviolet light, and subsequently purified using QIAquick Gel Extraction kit (Qiagen). Fragments were quantified using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). Fragments containing complementary USER junctions were added in an equimolar ratio of between 0.2 and 1 pmol to a 10 µl reaction containing 1 µl CutSmart Buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 µg/mL BSA at pH 7.9; New England Biolabs), 0.75 µL DpnI (New England Biolabs), and 0.75 µL USER enzyme (Uracil-DNA Glycosylase and DNA-glycosylase-lyase Endonuclease VIII; New England Biolabs). Reactions were incubated at 37 °C for 20 min, then heated to 80 °C for 3 min, and slowly cooled to 12 °C at 0.1 °C/s. During this assembly, uracil DNA-glycosylase catalyzes the excision of a dU, creating an apyrimidinic site at which Endonuclease VIII breaks the phosphodiester backbone. Assembled constructs were added to 100 µL 2x KCM (100 mM KCl, 30 mM CaCl₂, 50 mM MgCl₂ in MilliQ H₂O) and 100 µL competent cells. For all cloning, we used either Mach1F (Mach1 T1^R cells (Thermo Fisher Scientific) mated with F’ episome of S2060 strain³¹), NEB Turbo (New England Biolabs), DH5α (Thermo Fisher Scientific), or 10-beta (New England Biolabs) cells. Cells were flicked to mix and incubated on ice for 10 min, heat shocked at 42 °C for 1.5 min, and then placed back on ice for 2 min. Cells were allowed to recover in 1 mL 2xYT at 37 °C with shaking between 230 and 300 × g for at least 45 min. Cells were then streaked on 1.5% agar-2XYT supplemented with appropriate antibiotics and incubated at 37 °C for 16−18 h.

Transformation of chemically competent cells

To transform cells, 100 μL of competent cells were thawed on ice. To this, plasmid (2 μL each of miniprep-quality plasmid; up to two plasmids per transformation) and 100 μL KCM solution (100 mM KCl, 30 mM CaCl₂, and 50 mM MgCl₂ in H₂O) were added and flicked to mix. For transformations of greater than two plasmids, 2 µL of each plasmid was added to 30 µL competent cell/KCM mix. The mixture was incubated on ice for 10 min and heat-shocked at 42 °C for 90 s. The mixture was chilled on ice for 4 min, then 1 mL of 2XYT media was added. Cells were allowed to recover at 37 °C with shaking at 230 × g for at least 45 min, streaked on 2XYT media + 1.5% agar plates containing the appropriate antibiotics, and incubated at 37 °C for 16−18 h.

Selection criteria for representative E. coli tRNAs scaffolds

In cases where quadruplet-decoding tRNAs had been previously engineered, evolved, or discovered as natural suppressors, the same tRNA scaffolds were used in our evolution studies. This criterion was met for four tRNAs (Gly, Leu, Ser, Thr). In cases where either only a single copy of the tRNA scaffold was present in the E. coli genome (Cys, Trp) or all copies are genotypically identical with exception of the anticodon (Asn, Asp, Glu, His, Ile, Lys, fMet, Phe, Tyr), our choice was constrained to a single genotype. This criterion was met for eleven tRNAs. In all other cases, we chose tRNA scaffolds that were found at the end of their respective operons⁸² or sufficiently separated from neighboring tRNAs in order to use tRNAs that are endogenously expressed at high levels, ensure that we would not inadvertently capture >1 tRNA during amplification and cloning into the tRNA expression plasmid, and limit amplification issues using primers that may anneal within the highly structured sequence of a neighboring tRNA. This criterion was met for the remaining six tRNA (Ala, Met, Pro, Gln, Arg, Val). tRNA genes were amplified directly from E. coli genomic DNA.

LacZ selections

To nominate positions in lacZ for amino-acid-specific selections, we first confirmed the dependence of key positions in lacZ on the identity of the incorporated amino acid. Oligos bearing degenerate triplet codons (NNN) at requisite positions in lacZ were used to generate libraries of the constitutive lacZ-expressing plasmids pAB191b using USER cloning. Chemically competent BW25113 ∆lacI (Strain JW0336-1; CGSC#: 8528) cells were transformed with the libraries, recovered for 2 h at 37 °C with shaking at 230 × g, then plated as a serial dilution series on M9 minimal medium plates with glucose 1.8% agar, 0.01% thiamine, 22 mM glucose, 40 µg/mL chloramphenicol) or lactose (1.8% agar, 0.01% thiamine, 11 mM lactose, 0.033% Bluo-Gal, 40 µg/mL chloramphenicol) and incubated at 37 °C for 24−72. Libraries all showed >100-fold coverage as gauged by transformation efficiency (>6400 total CFUs), and a comparison of the total (glucose) to LacZ + (lactose) transformants were used to inform amino acid dependence. For positions where the observed frequency agreed with the expected frequency of LacZ+ colonies, 16−32 unique colonies were picked and surveyed by Sanger sequencing at the randomized residue. Positions that showed triplet codons exclusive to a single amino acid were used for quadruplet codon-qtRNA coevolution studies. For these library-cross-library selections, the identical protocol was used to generate the lacZ codon libraries with the exception that the used oligos encoded fully degenerate quadruplet codons (NNNN) at the requisite positions. Following plating on glucose, transformed colonies were used to make competent cells, which were later transformed with qtRNA libraries bearing fully degenerate anticodons (NNNN). Co-transformation efficiencies corresponded to >100-fold in all cases (>6.6E6 total CFUs). Single clone sequencing at the codon (lacZ) and anticodon (qtRNA) showed the identical sequences in most cases for colonies picked from lactose plates.

Measurement of quadruplet codon translation efficiency using luciferase reporter

S3489 cells were transformed with the luciferase-based activity reporter and qtRNA expression plasmids. Overnight cultures of single colonies grown in DRM media supplemented with maintenance antibiotics were diluted 500-fold into DRM media with maintenance antibiotics in a 96-well 2 mL deep well plate, with or without IPTG. The plate was sealed with a porous sealing film and grown at 37 °C with shaking at 900 × g. After 1 h, 175 μL of cells were transferred to a 96-well black-walled clear-bottom plate, and then 600 nm absorbance and luminescence were read using a CLARIOstar plate reader (Reader Control 5.21 R2, BMG Labtech) over the course of 8 h, during which the cultures were incubated at 37 °C. IPTG inducer concentration was 1 mM IPTG. For expression of orthogonal rRNA, the aTc inducer concentration was 100 ng/mL. GraphPad Prism (version 8) was used for plotting and data analysis, including calculation of means and standard deviations.

Calculation of percent wild-type luciferase (η)

In order to robustly compare toxic and non-toxic qtRNAs, all luminescence values are considered at OD₆₀₀ = 0.5 to account for differential growth rates. The percent of triplet codon translation efficiency, η, is calculated using the formula:

Where TriLux is the luminescence of the positive control, a luciferase encoded entirely with triplet codons; QuadLux_{qtRNA induced} is the luminescence produced by the quadruplet codon-bearing reporter upon qtRNA expression (1 mM IPTG); QuadLux_{qtRNA uninduced} is the luminescence produced by the quadruplet codon-bearing reporter upon qtRNA expression (0 mM IPTG).

Doubling time analyses

Colonies transformed with the appropriate wild-type tRNA, qtRNA, or a combination therein were picked and grown in DRM containing maintenance antibiotics. Following overnight growth at 37 °C with shaking at 900 × g, cultures were back diluted 100-fold into DRM containing maintenance antibiotics +/− IPTG. After growing for 1 h at 37 °C with shaking at 900 × g in a 96 deep well plate, 175 µl of each culture were transferred to a 96-well black wall, the clear bottom plate (Costar), and OD₆₀₀ was measured every 10 min over 10 h. The doubling time of wild type and qtRNA cultures were calculated using the Growthcurver package (version 0.3.1)⁸³ in R (version 4.0.3).

Fluorescence assays

S3489 cells were transformed with the sfGFP and/or mCherry-based activity reporter and qtRNA expression plasmid(s). For assays containing two plasmids (one qtRNA expression plasmid and one reporter plasmid), colonies were picked directly into DRM media supplemented with maintenance antibiotics (with or without 1 mM IPTG inducer) and allowed to grow overnight. For assays containing greater than two plasmids, overnight cultures of single colonies grown in DRM media supplemented with maintenance antibiotics were diluted 500-fold into DRM media with maintenance antibiotics in a 96-well 2 mL deep well plate (with or without 1 mM IPTG inducer). For three plasmid assays, the concentration of each antibiotic was cut by one third (i.e., carbenicillin, 16.7 μg/mL) and for four plasmid assays, the concentration of each antibiotic was cut by one fourth (i.e., carbenicillin 12.5 µg/mL). In all cases, the deep well plates were sealed with a porous sealing film and grown at 37 °C with shaking at 230 × g for 24−36 h. 150 μL of cells were transferred to a 96-well black-walled clear-bottom plate, and then 600 nm absorbance and fluorescence (sfGFP: λEx = 485 nm and λEm = 510 nm; mCherry: λEx = 587 nm and λEm = 610 nm) were read at 37 °C using a Spark (Tecan) plate reader running SparkControl v2.3. GraphPad Prism (version 8) was used for plotting and data analysis.

Calculation of percent wild-type sfGFP

After blank media subtraction, the percent of sfGFP triplet codon translation efficiency is calculated using the formula:

Where wild-type refers to the positive control sfGFP containing no quadruplet codons; Quad_sfGFP refers to fluorescence produced by the quadruplet codon-bearing reporter upon qtRNA expression (1 mM IPTG); OD600 and sfGFP values are normalized to blank media first. Threshold calculations refer to the average of fluorescence produced by the quadruplet codon-bearing reporter upon qtRNA expression (0 mM IPTG).

Sample preparation for quantification of qtRNA charging using mass spectrometry

Each qtRNA was co-expressed with C-terminal 6xHis-tagged sfGFP with the appropriate quadruplet codon replacing permissive residue Y151⁵⁶ in S3489 cells. Bacterial cultures between 4 and 50 mL were grown for 36 h at 37 °C in DRM media containing IPTG inducer and appropriate antibiotics. Cultures were then pelleted and frozen at −80 °C for at least 1 day. Once thawed and weighed, appropriate volumes of cOmplete, EDTA-free Protease Inhibitor Cocktail (1 tablet per 50 mL extraction solution; Millipore Sigma) and B-PER Bacterial Protein Extraction Reagent (4 mL per gram pellet; Thermo Fisher Scientific) were added to the cell pellet and gently pipetted up and down till homogenous. Samples were incubated for 1 h rotating at room temperature and centrifuged at 16,000 × RCF for 20 min to separate soluble proteins (supernatant from insoluble proteins (pellet). Soluble proteins were purified using either Ni-NTA spin column (Qiagen) or His-Spin Protein Miniprep (Zymo Research) and eluted in 150 µL. Resultant purified His-tagged proteins were denatured for 5 min at 95 °C, and 22 µL sample was mixed with 7.5 µL 4x NuPAGE dye and 0.5 µL 1 M DTT. The resulting samples and Blue Prestained Protein Standard (New England Biolabs) were run on a 12% Bis-Tris PAGE gel (Invitrogen) at 200 mA, for 15 min at 90 V and then 35 min at 200 V, using 1x NuPAGE MES SDS Running Buffer (Thermo Fisher Scientific). The SDS-PAGE gel was then washed in DI H₂O for 5 min three times, stained for 2 h in GelCode Blue Stain Reagent (Thermo Fisher Scientific), and destained in 50% methanol/water overnight. GelCode Blue stained SDS–PAGE gel lanes were then cut into ~2 mm squares, washed once more with 47.5/47.5/5 % methanol/water/acetic acid for 2 h, dehydrated with acetonitrile, and dried in a speed-vac. Reduction of disulfide bonds was then carried out by the addition of 30 μl 10 mM dithiothreitol (DTT) in 100 mM ammonium bicarbonate for 30 min. The resulting free cysteine residues were subjected to an alkylation reaction by removal of the DTT solution and the addition of 100 mM iodoacetamide in 100 mM ammonium bicarbonate for 30 min to form carbamidomethyl cysteine. These were then sequentially washed with aliquots of acetonitrile, 100 mM ammonium bicarbonate, and acetonitrile and dried in a speed-vac. The bands were enzymatically digested by the addition of 300 ng of trypsin (or chymotrypsin for arginine or lysine qtRNAs) in 50 mM ammonium bicarbonate to the dried gel pieces for 10 min on ice. Depending on the volume of acrylamide, excess ammonium bicarbonate was removed or enough was added to rehydrate the gel pieces. These were allowed to digest overnight at 37 °C with gentle shaking. The resulting peptides were extracted by the addition of 50 μL (or more if needed to produce supernatant) of 50 mM ammonium bicarbonate with gentle shaking for 10 min. The supernatant from this was collected in a 0.5 mL conical autosampler vial. Two subsequent additions of 47.5/47/5/5 acetonitrile/water/formic acid with gentle shaking for 10 min were performed with the supernatant added to the 0.5 mL autosampler vial. The organic solvent was removed, and the volumes were reduced to 15 μL using a speed vac for subsequent analyses.

Chromatographic separations

The digestion extracts were analyzed by reverse-phase high-performance liquid chromatography (HPLC) using Waters NanoAcquity pumps and autosampler and a ThermoFisher Orbitrap Elite mass spectrometer using a nano flow configuration. A 20 mm × 180 μm column packed with 5 μm Symmetry C18 material (Waters) using a flow rate of 15 μl per min for 3 min was used to trap and wash peptides. These were then eluted onto the analytical column which was self-packed with 3.6 μm Aeris C18 material (Phenomenex) in a fritted 20 cm × 75 μm fused silica tubing pulled to a 5 μm tip. Elution was carried out with a gradient of isocratic 1% Buffer A (1% formic acid in H₂O) for 1 min (250 nL min⁻¹), followed by increasing Buffer B (1% formic acid in acetonitrile) concentrations to 15% B at 20.5 min, 27% B at 31 min and 40% B at 36 min. The column was washed with high percent B and re-equilibrated between analytical runs for a total cycle time of approximately 53 min.

Mass spectrometry

The mass spectrometer was operated in a dependent data acquisition mode where the 10 most abundant peptides detected in the Orbitrap Elite (ThermoFisher) using full scan mode with a resolution of 240,000 were subjected to daughter ion fragmentation in the linear ion trap. A running list of parent ions was tabulated to an exclusion list to increase the number of peptides analyzed throughout the chromatographic run. The resulting fragmentation spectra were correlated against custom databases using PEAKS Studio X (Bioinformatics Solutions). To calculate the limit of detection and relative amino acid abundance, the results were matched to a library of GFP variants with each of the 20 canonical amino acids at respective residues. The abundance of each species was quantified by calculating the area under the curve of the ion chromatogram for each peptide precursor. The limit of detection was 10⁴ (arbitrary units), the lower limit for area under the curve for a peptide on this instrument.

Phage supernatant filtration

To filter 500 μL of phage, bacteria were pelleted by centrifugation at 8,000 × RCF for 2 min in a tabletop centrifuge. The supernatant was transferred to a 0.22 μm filter column and centrifuged at 1000 × RCF for 1 min to create filtered phage flow-through. To filter 50 mL of phage supernatant, 50 mL of culture was similarly pelleted. The supernatant was applied to a Steriflip (Millipore Sigma) 0.22 μm vacuum filter unit. To filter up to 150 μL of phage in 96-well plate format, the 96-well plate of bacteria was pelleted by centrifugation at 1,000 × RCF for 10 min. 150 μL of supernatant was applied to wells of a 96-well 0.22 μm filter plate taped atop a 96-well PCR plate and centrifuged at 1000 × RCF for 1 min to create filtered phage flow-through. Phage can be stored at 4 °C in 96-well plate format covered with an aluminum sealing film. For frequently accessed phage samples, we recommend storage in 2 mL screw-cap tubes to minimize potential phage contamination generated from opening snap-caps.

Standard phage cloning

Competent E. coli S3489 cells were prepared (as described) containing pJC175e, a plasmid expressing pIII under control of the phage shock promoter⁸⁴. To clone ΔpIII M13 bacteriophage, PCR fragments were assembled using USER, as above. The annealed fragments were transformed into competent S3489-pJC175e competent cells (as described), which complement pIII deletion from the bacteriophage. Transformants were recovered in 2xYT media overnight, shaking at 230 × g at 37 °C. The phage supernatant from the resulting culture was filtered (as described), and plaqued (as described). Clonal plaques were expanded overnight, filtered, and Sanger sequenced.

Phage library cloning

We do not recommend USER cloning for library creation inside of high-secondary structure tRNAs; instead, we used degenerate primers and blunt-end ligation. Primers were designed containing a NNNN degenerate anticodon. To reduce nucleotide bias during blunt end ligation assembly, the last degenerate base was designed to be at least one base away from the end of the primer. For each library, 200 μL of PCR product was used. The entirety of this PCR product was run on a gel, extracted, and purified using spin column purification. Background plasmid was digested using DpnI (New England Biolabs), and the remaining PCR product was purified again using spin columns, and ligated. The ligation product was transformed into competent E. coli S3489 cells containing pJC175e. Transformants were recovered in 2xYT media overnight, shaking at 230 × g at 37 °C. The phage supernatant from the resulting culture was filtered and plaqued.

Manual phage plaque assays

S3489 cells were transformed with the Accessory Plasmid of interest. Overnight cultures of single colonies grown in 2xYT media supplemented with maintenance antibiotics were diluted 1,000-fold into fresh 2xYT media with maintenance antibiotics and grown at 37 °C with shaking at 230 × g to OD₆₀₀ ~0.6–0.8 before use. Bacteriophage were serially diluted 100-fold (four dilutions total) in H₂O. 100 μL of cells were added to 100 μL of each phage dilution, and to this 0.85 mL of liquid (70 °C) top agar (2xYT media + 0.6% agar) supplemented with 2% Bluo-Gal was added and mixed by pipetting up and down once. This mixture was then immediately pipetted onto one quadrant of a quartered Petri dish already containing 2 mL of solidified bottom agar (2XYT media + 1.5% agar). After solidification of the top agar, plates were incubated at 37 °C for 16–18 h.

Phage enrichment assays

S3489 cells were transformed with the Accessory Plasmids of interest as described above. Overnight cultures of single colonies grown in 2XYT media supplemented with maintenance antibiotics were diluted 1,000-fold into DRM media with maintenance antibiotics and grown at 37 °C with shaking at 230 × g to OD₆₀₀ ~0.4–0.6. Cells were then infected with bacteriophage at a starting titer of 10⁵ pfu/mL. Cells were incubated for another 16–18 h at 37 °C with shaking at 230 × g. Supernatant was filtered and stored at 4 °C. The phage titer of these samples was measured in an activity-independent manner using a plaque assay containing E. coli bearing pJC175e.

Continuous flow PACE

Unless otherwise noted, PACE apparatus, including host cell strains, lagoons, chemostats, and media, were all used as previously described⁶⁶. Chemically competent S3489 cells were transformed with the Accessory Plasmid and the mutagenesis plasmid (MP) MP6³⁵ as described above, plated on 2xYT media + 1.5% agar supplemented with 25 mM glucose (to prevent induction of mutagenesis) in addition to maintenance antibiotics, and grown at 37 °C for 18–20 h. To validate MP functionality prior to evolution, four colonies were picked into 10 μL DRM media and diluted 10-fold six times; these dilutions were plated on either agar plates with maintenance antibiotics and 25 mM glucose, or 10 mM arabinose; we expected that colonies plated on arabinose would be of reduced size when the MP is functional. The remainder of the dilutions were added to 2 mL DRM media with maintenance antibiotics, grown at 37 °C with shaking until they reached OD₆₀₀ ~0.4–0.8, and then used to inoculate a turbidostat containing 30 mL DRM media. The turbidostat maintained the growing culture at OD₆₀₀ ~0.7–0.8. Prior to bacteriophage infection, lagoons were continuously diluted with culture from the turbidostat at 1 lagoon vol/h and pre-induced with 10 mM arabinose for at least 45 min to induce mutagenesis. Samples (500 μL) of the SP population were taken at indicated times from lagoon waste lines. These were centrifuged at 8,000 RCF for 2 min, and the supernatant was passed through a 0.22 μm filter and stored at 4 °C. Lagoon titers were determined by plaque assays using S3489 cells transformed with pJC175e.

Aminoacyl-tRNA synthetase expression and purification

E. coli SerRS, ArgRS, and TyrRS were overexpressed in BL21 (DE3) E. coli cells and purified as previously described³⁵ with slight modifications. Cells were grown at 37 °C until OD₆₀₀ 0.6 and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside for 3 h at 30 °C. Cells were resuspended in Buffer A (50 mM HEPES-KOH [pH 7.5], 300 mM NaCl, 10 mM β-mercaptoethanol, 3 mM MgCl₂, 10 mM imidazole) along with a protease inhibitor tablet (Roche, cOmplete Mini, EDTA-free) and subjected to sonication. The lysate was centrifuged at 38,000 × g for 40 min at 4 °C and the synthetases were purified via nickel affinity chromatography. The synthetases were eluted with Buffer B (50 mM HEPES-KOH [pH 7.5], 300 mM NaCl, 10 mM β-mercaptoethanol, 3 mM MgCl₂, 250 mM imidazole) and incubated with His-tagged TEV protease for 1 h at 37 °C. The aaRS-TEV protease solution was dialyzed into Buffer A, subjected to nickel affinity chromatography to isolate the aaRS, dialyzed into a storage buffer (50 mM HEPES-KOH [pH 7.5], 100 mM NaCl, 10 mM β-mercaptoethanol, 3 mM MgCl₂, 50% glycerol), and stored at −80 °C.

In vitro aminoacylation assay

All qtRNAs were in vitro transcribed using T7 RNA polymerase and gel purified as previously described³⁵. Prior to use, the qtRNAs were heated to 85 °C and slowly cooled down to room temperature in the presence of 10 mM MgCl₂ to allow proper refolding. In vitro aminoacylation of tRNA^Ser_UAGA by E. coli seryl-tRNA synthetase, tRNA^Arg_UAGA by E. coli arginyl-tRNA synthetase, and tRNA^Tyr_UAGA by E. coli tyrosyl-tRNA synthetase were performed as previously described⁶⁵. Briefly, reactions contained 50 mM HEPES-KOH [pH 7.3], 4 mM ATP, 25 mM MgCl₂, 0.1 mg/mL bovine serum albumin, 20 mM KCl, 20 mM 2-mercaptoethanol, 4 µM qtRNA, amino acid (25 µM L-[¹⁴C]-Ser, 6 µM L-Arg (2 µM L-[¹⁴C]-Arg, 4 µM L-Arg), or 6 µM L-Tyr (2 µM L-[¹⁴C]-Tyr, 4 µM L-Tyr)) and E. coli aaRS (50 mM SerRS, 30 nM ArgRS, or 30 nM TyrRS). The reactions were incubated at 37 °C and 8 µL aliquots were removed at given intervals, spotted onto 3 MM filter papers (presoaked with 5% trichloroacetic acid and dried), immersed in 5% TCA to precipitate aminoacyl-qtRNAs, and then subjected to scintillation counting.

Diagrams and Crystal Structures

R2R (version 1.0.6) was used to generate tRNA diagrams. R2R is free software available from https://sourceforge.net/projects/weinberg-r2r/. The co-crystal structure of E. coli tRNA-Gln and glutaminyl-tRNA synthetase was visualized in PyMOL version 2.5.0.

Reporting summary

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