Biochemical Assays

The assay conditions of EcDTD and PfDTD with various substrates have been described in [15]. A representative thin-layer chromatographic run is given in S8 Fig. E. coli tRNA^Gly was charged with glycine in a reaction mix containing 100 mM HEPES pH 7.2, 30 mM KCl, 10 mM MgCl₂, 2 mM ATP, 50 mM glycine, 1 μM tRNA^Gly, and 2.2 μM T. thermophilus glycyl-tRNA synthetase (UniProt ID: {"type":"entrez-protein","attrs":{"text":"P56206","term_id":"62296700"}}P56206) incubated at 37°C for 15 min. E. coli L-Ala-tRNA^Ala was generated by the protocol given in [25]. DTD deacylation kinetics on Gly-tRNA^Gly was performed following the method described in [25] and [27].

EF-Tu activation was done by incubating 10 μM T. thermophilus EF-Tu in a solution containing 50 mM HEPES pH 7.2, 20 mM MgCl₂, 250 mM NH₄Cl, 5 mM DTT, 2 mM GTP, 3 mM phosphoenol pyruvate, and 50 μg ml^-1 pyruvate kinase at 4°C for 3 h. Competition (deacylation) assays were performed in a solution of 100 mM HEPES pH 7.2 and 2.5 mM DTT containing 2 μM activated EF-Tu, 200 nM Gly-tRNA^Gly and varying concentrations of EcDTD.

3′-end modifications of E. coli tRNA^Gly were generated using in vitro-transcribed tRNA^Gly-CC lacking the terminal adenosine. Ten μM tRNA^Gly-CC was incubated with [α-³²P]-2′-dATP, 2.6 μM CCA-adding enzyme (UniProt ID: {"type":"entrez-protein","attrs":{"text":"P06961","term_id":"115939"}}P06961), 50 mM Tris pH 7.6, 20 mM MgCl₂, 0.5 mM DTT, and 0.6 mM CTP at 37°C for 4 h to generate tRNA^Gly(2′-dA76). tRNA^Gly(2′-FdA76) was similarly produced using 400 μM 2′-FdATP (Jena Biosciences) instead of [α-³²P]-2′-dATP; however, tRNA^Gly-CC used for making tRNA^Gly(2′-FdA76) was body-labeled by performing in vitro transcription with [α-³²P]-UTP. Aminoacyltion of the 3′-end modified tRNAs with glycine was done using the conditions given for wild-type tRNA charging. Time-point–based deacylation assays with Gly-tRNA^Gly(2′-dA76) were performed using thin-layer chromatography–based method, whereas those with Gly-tRNA^Gly(2′-FdA76) were done using acid urea polyacrylamide gel electrophoresis [67]. Each point on the deacylation curves represents the mean of three independent readings; the error bars represent one standard deviation from the mean.