Production of labelled tRNAArg

tRNA was produced following the protocol by Avcilar-Kucukogze et al. (40). In brief, for in vitro transcribed tRNA two overlapping single-stranded oligonucleotides encoding the 5′ end sequence of the sense strand with an upstream T7 RNA polymerase promoter sequence and the 3′ end sequence of the antisense strand were annealed and filled using the Large Klenow Fragment of DNA polymerase. The resulting double-stranded DNA template was in vitro transcribed using T7 RNA polymerase followed by incubation with DNase I to digest the DNA template. The tRNA was purified by excision from 6% polyacrylamide gel electrophoresis (PAGE). Excised bands were incubated in crush and soak buffer (200 mM NaCl, 1 mM EDTA, 10 mM HEPES, pH 7.5), filtered through glass wool and tRNA was extracted by ethanol precipitation. For the over-production of tRNA^Arg in P. canavaninivorans, the tRNA^Arg sequence was inserted into the rhamnose-inducible expression vector pJeM1 (41) (Addgene #135088, SI Item 1)). P. canavaninivorans was transformed with the resulting plasmid by electroporation and transformants were selected by kanamycin resistance. A single colony was pre-cultured overnight, diluted 1:100 and grown at 30°C to an OD₆₀₀ of 0.4. Then, rhamnose was added to induce tRNA production and the culture was grown for approximately 16 h. Cells were collected by centrifugation and tRNA was extracted as described by Avcilar-Kucukogze et al. (40). After extraction, the total tRNAs enriched in tRNA^Arg were also further purified by 6% PAGE. tRNA was 3′ α-³²P-ATP labelled using purified E. coli cca tRNA nucleotidyltransferase (overexpression clone from the ASKA collection, (42)), following the protocol by Evans et al. (43). In summary, tRNA was first refolded by heating and slow cooling to ensure the right tRNA conformation and then treated with cca adding enzyme in a reaction where the equilibrium is shifted to exchange the terminal adenosine with α-³²P-ATP by the addition of inorganic phosphatase and CTP. For the production of 5′ γ-³²P labelled tRNA, unlabelled purified tRNA was first dephosphorylated using shrimp alkaline phosphatase and then labelled with γ-³²P-ATP using T4 polynucleotide kinase following the manufacturer's instructions. In both cases labelled tRNA was purified by PAGE.