Translational roles of the C75 2′OH in an in vitro tRNA transcript at the ribosomal A, P and E sites

Aminoacyl-tRNAs containing a deoxy substitution in the penultimate nucleotide (C75 2′OH → 2′H) have been widely used in translation for incorporation of unnatural amino acids (AAs). However, this supposedly innocuous modification surprisingly increased peptidyl-tRNAAla ugc drop off in biochemical assays of successive incorporations. Here we predict the function of this tRNA 2′OH in the ribosomal A, P and E sites using recent co-crystal structures of ribosomes and tRNA substrates and test these structure-function models by systematic kinetics analyses. Unexpectedly, the C75 2′H did not affect A- to P-site translocation nor peptidyl donor activity of tRNAAla ugc. Rather, the peptidyl acceptor activity of the A-site Ala-tRNAAla ugc and the translocation of the P-site deacylated tRNAAla ugc to the E site were impeded. Delivery by EF-Tu was not significantly affected. This broadens our view of the roles of 2′OH groups in tRNAs in translation.

. Interactions of the tRNA C75 2′OH in ribosomal A-, P-and E-sites in crystal structures. (a) tRNA substrates at the aminoacyl (green), peptidyl (yellow) and exit (red) sites on the 70S ribosome in 3 dimensions. The PTC is circled. (b) Two-dimensional chemical structures showing the covalent bonds separating the C75 2′OH groups (circled) from the nucleophile and electrophile of the peptidyl transfer reaction (blue arrow). (c-e) Crystal structures of the A-, P-and E-site tRNA substrates, respectively. Models were constructed with UCSF Chimera and PDB file 1VY4 18 . In the original PDB file, three water molecules found within the PTC were proposed to be essential for forming a "proton wire" to couple AA-tRNA accommodation and peptide bond formation. Two of these water molecules are ringed with white dashed circles and labeled W1 and W2 (W3 is not visible in this view). H-bonds are shown with white thin lines. (c) A-site Phe-tRNA Phe in green, with the 2′OH in magenta. C75 forms a Watson-Crick base pair with G2553 of 23S rRNA, and the 2′OH of C75 forms an H bond with the O4 of the A76 sugar pucker. N indicates that Phe was linked to the tRNA with an amide bond. (d) P-site fMet-tRNA fMet in yellow, with the 2′OH in magenta. C75 forms a Watson-Crick base pair with G2251 of 23S rRNA, and the 2′OH of C75 forms H bonds with W2 and the 2′OH of ribose C2073 of 23S rRNA. N indicates that fMet was linked to the tRNA with an amide bond. (e) E-site tRNA Phe in red, with the 2′OH in magenta. C75 does not form a base pair, whereas the 2′OH of C75 forms an H-bond with the N7 of the base of A76 in the same tRNA.
Scientific REPORTS | 7: 6709 | DOI: 10.1038/s41598-017-06991-6 The C75 2′H impedes the peptidyl acceptor activity of Ala-tRNA Ala but not delivery by EF-Tu. To test our predictions based on crystal structures, we prepared Ala-tRNA Ala substrates with either C75 Figure 1). Ala-tRNA _enzymatic ugc AlaB was prepared from in vitro transcribed full-length tRNA Ala by charging with Ala using the AA-tRNA synthetase AlaRS. Ala-tRNA _dC ugc AlaB was prepared by ligating in vitro-transcribed 3′CA-truncated tRNA Ala to N-NVOC-Ala-pdCpA followed by photolytic removal of the NVOC group. We measured the following successive individual translation steps, listed in the order in which they occur in translation (Fig. 2): GTP hydrolysis on EF-Tu (to assay delivery of Ala-tRNA Ala to the ribosomal A site), the subsequent time for fMet-Ala (fMA) dipeptide bond formation (to assay peptidyl acceptor activity of Ala-tRNA Ala ), A to P site translocation of fMA-tRNA Ala , fMet-Ala-Phe (fMAF) tripeptide formation from EF-Tu:GTP:Phe-tRNA Phe and fMA-tRNA Ala (to assay peptidyl donor activity of P site fMA-tRNA Ala ), and P to E site translocation of tRNA Ala .
Initially, we measured the rates of fMA dipeptide formation at different EF-Tu:GTP:Ala-tRNA Ala ternary complex concentrations of the two kinds of substrates (Supplementary Table 1). Fitting these rates to a hyperbolic Michaelis-Menten equation allowed the estimation of the maximal overall rate of fMA dipeptide formation, k cat , and the K M values ( Fig. 3a and Table 1). k cat for Ala-tRNA _dC ugc AlaB was 59 ± 4 s −1 whereas for Ala-tRNA _enzymatic ugc AlaB it was significantly faster at 157 ± 37 s −1 , but the respective K M values were similar (3.5 ± 0.5 μM and 3.8 ± 1.4 μM). This indicated that one or more steps up to the peptidyl transfer reaction were impeded by replacing the C75 2′OH with the 2′H. Next, we measured the mean times for GTP hydrolysis on EF-Tu (τ GTP ) and dipeptide formation (τ dip ) simultaneously (Fig. 3b,c and Table 1). τ GTP for the two cases was the same, ~8 msec, so the delivery of the AA-tRNA to the ribosomal A site by EF-Tu:GTP was not impeded by the lack of the C75 2′OH. However, the time for dipeptide formation, τ dip , with Ala-tRNA _dC ugc AlaB (~33 msec) was about two-fold longer than that of Ala-tRNA _enzymatic ugc AlaB (~18 msec). This indicated impeded (i) release of the 3′CCA end of the tRNA from EF-Tu:GDP, (ii) AA-tRNA accommodation, and/or (iii) peptidyl transfer reaction. The overall mean time for these three steps, τ acc,pep , could be calculated by subtracting τ GTP from τ dip ( Table 1), but independently measuring these three steps is not tractable. Although an impeded release by EF-Tu-GDP or accommodation step cannot be ruled out, we speculate that it is the peptidyl transfer reaction that is affected by the C75 2′H. The conformation of peptidyl transferase center is induced by base pairing of the A-site tRNA penultimate C with the G2553 of the 23S rRNA, and proper orienting of the ester link of the P-site tRNA is important for peptidyl transfer [19][20][21][22][23][24] . Replacing the penultimate C with dC might change the ribonucleotide-favored C3′-endo sugar conformation to a deoxyribonucleotide-favored C2′-endo sugar conformation in the tRNA 25 . The result also supports our prediction that the loss of the A-site H bond to the O4 of the A76 sugar, which in turn is attached to the AA nucleophile, may affect correct positioning for nucleophilic attack.
The C75 2′H does not affect A-to P-site translocation nor peptidyl donor activity. We next analyzed for effects on translocation of fMA-tRNA Ala from the A to P site and the subsequent donor activity of the dipeptidyl-tRNA in peptidyl transfer. Experimentally, 3 reactions could be measured in parallel 26 (Fig. 4a): (i) fMA formation from fMet-tRNA fMet and Ala-tRNA Ala (step1); (ii) fMAF tripeptide formation from fMet-tRNA fMet , Ala-tRNA Ala and Phe-tRNA Phe (tot); and (iii) fMAF tripeptide formation from translocated fMA-tRNA Ala and Phe-tRNA Phe (step3). The mean time for translocation from A site to P site could then be calculated as Fig. 4 and Table 2). The τ step1 for reaction with Ala-tRNA _dC ugc AlaB (~29 msec) was larger than that when the tRNA was fully ribo-tRNA Ala (~16 msec), as expected. Surprisingly, the τ step3 for the two cases . Schematic illustration of the kinetic steps in tripeptide synthesis. The EF-Tu:GTP:Ala-tRNA Ala ternary complex was added to the 70S initiation complex and the mean time for GTP hydrolysis on EF-Tu was measured. After GTP hydrolysis and ET-Tu:GDP was dissociated from the ribosome, the Ala-tRNA Ala was accommodated to accept the fMet to form the fMA dipeptide. The mean time for fMA-tRNA Ala formation was measured. After the formation of fMA-tRNA Ala , this dipeptidyl-tRNA was translocated from the A site to the P site by EF-G and could then donate the fMA dipeptide to the incoming A site Phe-tRNA Phe to form the fMAF tripeptide. The resulting P site deacylated tRNA Ala was translocated to the E site along with the peptidyl-tRNA Phe translocating to the P site. had no significant difference ( Table 2), indicating that the C75 2′H did not affect the peptidyl donor activity of the P site fMA-tRNA Ala in the peptidyl transfer to Phe-tRNA Phe . Also unexpectedly, the calculated τ step2 for the two cases were similar, indicating that the penultimate dC did not affect translocation of fMA-tRNA Ala from the ribosomal A site to the P site.  Table 1). Experiments were conducted at least in duplicates.
Scientific REPORTS | 7: 6709 | DOI:10.1038/s41598-017-06991-6 The C75 2′H inhibits P-to E-site translocation. Finally we analyzed for effects on P-to E-site translocation of the P-site deacylated C75 2′H tRNA Ala . This step occurred 3 times during our incorporation of 5 consecutive Ala-tRNA Ala substrates 11 . In order to isolate the effect of the C75 2′H on a single translocation step, we used a simpler translocation assay here: monitoring the mRNA movement with a 3′ pyrene-labeled mMA 2 FT mRNA 27,28 . A post-translocation complex programmed with this fluorescent mRNA and fMA 2 -tRNA Ala in the P site (and the same tRNA Ala at the E site, if still bound) was rapidly mixed with EF-Tu:GTP:Phe-tRNA Phe ternary complex together with EF-G:GTP on a stopped-flow apparatus (Fig. 5a). First, before translocation can occur, there is a very fast peptide bond formation. Then the deacylated P-site tRNA Ala is translocated to the E site along with the mRNA movement. The monitored fluorescence signal is quenched with the 3′ pyrene label moving towards the ribosome (Fig. 5b) 27,28 . The overall mean time for the fluorescence change is the sum of the mean times for the Phe-tRNA Phe incorporation and EF-G-catalyzed mRNA movement. The overall mean time for Ala-tRNA _enzymatic ugc AlaB was ~90 (91 ± 3) msec, compared with ~190 (189 ± 7) msec for Ala-tRNA _dC ugc AlaB . Assuming the incorporation mean time for Phe-tRNA Phe was the same as measured in the step3 described above (~15 msec for both the cases, Table 2), translocating the tRNA carrying the C75 2′H from P site to E site would take ~175 msec. This is ~2-fold slower compared with the P-to E-site translocation of the fully ribo-tRNA (~75 msec) and also τ step2 values for rC and dC (Table 2). Again, this effect was not predicted from the crystal structures (Fig. 1e).

Conclusion
By systematic kinetic analyses, we found that the C75 2′H in the A-site Ala-tRNA Ala ugc impeded its peptidyl acceptor activity and the C75 2′H in the P-site deacylated tRNA Ala ugc slowed translocation to the E site. This defines the mechanism by which this subtle atomic mutation in the tRNA Ala ugc decreased the efficiencies of ribosomal polymerization. Delivery by EF-Tu was not significantly affected. Unexpectedly, at least according to our predictions from high-resolution co-crystal structures of full-length tRNA fMet and tRNA Phe analogs bound to the ribosome, the C75 2′H in tRNA Ala ugc did not affect peptidyl donor activity or A-to P-site translocation. This suggests that, either the interpretations of the bona fide snap shots of the translation machinery cannot directly be applied to the systems when different tRNA substrates are used, or our kinetics approach can lead to higher resolution of the AA-tRNA τ tot (msec) τ step1 (msec) τ step3 (msec) τ step2 (msec)  Table 2. Kinetic parameters for fMAF tripeptide formation (see Fig. 4). Mean values were calculated from at least two independent experimental results with their propagated standard deviations. mechanisms of translation and thus can serve as a good complementary technology to the structural methods. Generalization of our conclusions will require follow up studies with other tRNAs. Nevertheless, our kinetics results define the first translational roles of a C75 2′OH in an in vitro tRNA transcript and expand our understanding of the importance of 2′OH groups in RNA function.

Materials and Methods
Materials. N-NVOC-Ala-pdCpA was prepared and characterized as before 29 . Synthetic 3′CA-truncated and full-length tRNA ugc AlaB were prepared as before 10 . After ligation of the N-NVOC-Ala-pdCpA to the 3′CA-truncated tRNA AlaB transcript, N-NVOC-Ala-tRNA AlaB was purified on a Q-column as described and the NVOC protecting group was removed by photolysis 30 . Synthetic mRNA, mMAF, was prepared from in vitro transcription with DNA template made from synthetic oligos 31 and the sequence was 5′ gggaauucgggcccuuguuaacaauuaaggagguauaucauggcauuuuaauugcagaaaaaaaaaaaaa 3′ with the coding region in bold (the processivity of ribosomal polymerization using mRNA transcribed from synthetic oligos was indistinguishable from that using mRNA from cloned plasmid based on kinetics with mMAAAAA 11 ). The over-expressed E. coli tRNA fMet and tRNA Phe were prepared respectively as described 32,33 . All other reagents were prepared as described 34 . Simultaneous measurement of GTP hydrolysis and dipeptide synthesis. To simultaneously measure the mean times for GTP hydrolysis on EF-Tu (τ GTP ) and dipeptide formation (τ dip ), a ribosomal mix was prepared similarly to that described above, except that the 70S ribosome was added to 2 μM and [ 3 H] fMet-tRNA fMet to 2.5 μM. In the ternary complex mix, unlabeled GTP was omitted and ATP was added to 2 mM. The ternary complex mix contained 1 μM . Sample treatment and data analysis were done in the same way as described 31 . The overall mean time, τ acc,pep , for all the steps that lead to peptide bond formation after GTP hydrolysis on EF-Tu was obtained by subtracting the mean time for GTP hydrolysis on EF-Tu from the mean time for dipeptide formation.

Kinetics of dipeptide synthesis at different ternary complex concentrations.
A-to P-site translocation assay. To measure the rate of the translocation step between the formation of fMA dipeptide and the formation of fMet-Ala-Phe (fMAF) tripeptide, two reactions were done in parallel 27 . The first reaction was fMAF tripeptide synthesis starting from 70S initiation complex such that the mean times for fMA dipeptide formation (τ step1 ) and fMAF tripeptide formation (τ tot ) could be deduced. In this reaction, the ribosomal mix contained 0.6 μM 70S ribosome, 0.9 μM IF1, 0.3 μM IF2, 0.9 μM IF3, 1 μM mMAF and 0.5 μM AlaB . The reaction and sample treatment were performed in the same way as described above for the dipeptide synthesis experiment. In the second reaction, fMAF tripeptide was synthesized from the posttranslocation complex that had the fMA-tRNA in the P site, such that the second peptide bond formation mean time, τ step3 , could be obtained. In this reaction, three mixtures were prepared. The ribosomal mix had doubled concentrations of the components as in the ribosomal mix in the first reaction. An elongation mix, E1, contained 8 μM EF-Tu, 1 μM EF-Ts, 4 μM EF-G and 12 μM Ala-tRNA _dC ugc AlaB or 0.4 mM alanine, 0.4 unit/μL AlaRS and 12 μM full-length transcript tRNA ugc AlaB . The third mix, E2, contained 16 μM EF-Tu, 1.5 μM EF-Ts, 20 μM EF-G, 0.4 mM phenylalanine, 0.4 unit/μL PheRS and 8 μM tRNA Phe . After preincubation for 15 min at 37 °C for the three mixtures, equal volumes of the ribosomal mix and E1 mix were rapidly mixed in the reaction tube for 5 sec before incubation on ice for 10 min. The resulting mixture and E2 mix were applied to the quench-flow apparatus for kinetic experiments. The reaction and sample treatment were performed in the same way as described above. Dipeptide, tripeptide and unreacted fMet was separated on C18 reverse phase HPLC by eluting with 0% methanol/99.9% H 2 O/0.1% trifluoroacetic acid for 20 min at 1 mL/min and another 10 min at 0.45 mL/min, followed by 30 min elution with 58% methanol/42% H 2 O/0.1% trifluoroacetic acid at 0.45 mL/min. Data analysis was done as described 26 and the complete translocation time, τ step2 , was calculated as τ step2 = τ tot − τ step1 − τ step3 . P-to E-site translocation rate measurement. The 3′ pyrene-labeled mMA 2 FT mRNA was chemically synthesized by IBA GmbH (Göttingen), kindly provided M. Holm and S. Sanyal, and had the sequence 5′ uaacaauaaggagguauuaaauggcagcauuuacg 3′. Three mixtures were prepared for this measurement similar to the three mixtures in the A-to P-site translocation assay. A ribosomal mix contained 0.8 μM 70S ribosome, 1.2 μM IF1, 0.4 μM IF2, 1.2 μM IF3, 1 μM [ 3 H]fMet-tRNA fMet and 1.5 μM pyrene-labeled mRNA. An elongation mix, E1, contained 2 μM EF-Tu, 0.25 μM EF-Ts, 4 μM EF-G and 2 μM Ala-tRNA _dC ugc AlaB or 0.2 mM alanine, 0.2 unit/μL AlaRS and 2 μM full-length transcript tRNA ugc AlaB . The third mix, E2, contained 10 μM EF-Tu, 0.5 μM EF-Ts, 38 μM EF-G, 0.4 mM phenylalanine, 0.4 unit/μL PheRS and 6 μM tRNA Phe . After preincubation of the three mixtures for 15 min at 37 °C, equal volumes of the ribosomal mix and E1 mix were rapidly mixed in the reaction tube for 10 sec before incubation on ice for 10 min. The resulting mixture and E2 mix were loaded onto a stopped flow instrument (Applied Photophysics SX20). Pyrene fluorescence was excited at 343 nm and the change in fluorescence after mixing was recorded using a 360 nm long pass filter (Newport 10CGA-360). Data analysis was done as described 27 .
Data availability. All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).