Lack of Discrimination Against Non-proteinogenic Amino Acid Norvaline by Elongation Factor Tu from Escherichia coli †

The GTP-bound form of elongation factor Tu (EF-Tu) brings aminoacylated tRNAs (aa-tRNA) to the A-site of the ribosome. EF-Tu binds all cognate elongator aa-tRNAs with highly similar affinities, and its weaker or tighter binding of misacylated tRNAs may discourage their participation in translation. Norvaline (Nva) is a non-proteinogenic amino acid that is activated and transferred to tRNA by leucyltRNA synthetase (LeuRS). No notable accumulation of Nva-tRNA has been observed in vitro, because of the efficient post-transfer hydrolytic editing activity of LeuRS. However, incorporation of norvaline into proteins in place of leucine does occur under certain conditions in vivo. Here we show that EF-Tu binds Nva-tRNA and Leu-tRNA with similar affinities, and that Nva-tRNA and Leu-tRNA dissociate from EF-Tu at comparable rates. The inability of EF-Tu to discriminate against norvaline may have driven evolution of highly efficient LeuRS editing as the main quality control mechanism against misincorporation of norvaline into proteins. (doi: 10.5562/cca2173)


INTRODUCTION
Bacterial elongation factor Tu (EF-Tu) delivers elongator aminoacyl-tRNAs (aa-tRNA) to the ribosome, where they are utilized in protein synthesis. 1,2EF-Tu belongs to a group of G-binding proteins that alternate between inactive and active forms by a mechanism involving the exchange of GDP with GTP.The GTPbound form (EF-Tu:GTP) binds all elongator aa-tRNAs with very similar affinities in the nanomolar range, [3][4][5] thus enabling a consistent rate of protein translation.7][8] Coupling of aa-tRNA binding by EF-Tu with its GTP/GDP cycle is important for accurate recognition of aa-tRNAs on the ribosome.Binding of the ternary EF-Tu:GTP:aa-tRNA complex to a cognate mRNA codon triggers the GTPase activity, which releases cognate aminoacylated tRNA for binding in the ribosomal A site. 2 Interestingly, EF-Tu:GTP displays substantial specificity for both the amino acid and the tRNA portions of its aa-tRNA ligands. 4,5,94][5] Thus, weak-binding amino acids are esterified to cognate tRNAs that bind EF-Tu tightly, while tight-binding amino acids are matched with tRNAs that bind EF-Tu weakly.Strong thermodynamic contribution of either the esterified amino acid or the tRNA body, therefore, compensates for the weak thermodynamic contribution of another portion of the cognate aa-tRNA pair.In contrast, tRNAs acylated with non-cognate amino acids (misacylated tRNAs) bind EF-Tu with a broad range of affinities, varying from 60-fold weaker to 120-fold tighter than cognate aa-tRNAs.][12] Aminoacyl-tRNA synthetases (aaRSs) covalently link amino acids with cognate tRNAs in a two-step synthetic reaction that proceeds via an aminoacyladenylate intermediate. 13Norvaline (Nva) is a nonproteinogenic amino acid that may increase in concentration to as high as 1 mmol dm -3 during unlimited growth of Escherichia coli on glucose after a down-shift in oxygen levels. 14Interestingly, a low but readily detectable incorporation of norvaline for leucine was observed in recombinant human hemoglobin produced in E. coli, and the extent of misincorporation strongly Croat.Chem.Acta 86 (2013) 73.correlated with the ratio of free norvaline to leucine. 15hus, under conditions that promote norvaline accumulation, leucyl-tRNA synthetase (LeuRS) catalyzes formation of Nva-tRNA Leu in vivo.Norvaline possesses a linear three-carbon side chain that cannot be excluded from the LeuRS amino acid binding site on steric grounds (Figure 1a), preventing efficient discrimination in the synthetic reactions alone.Therefore, to achieve the accuracy required for protein synthesis, LeuRS possesses intrinsic hydrolytic editing activities to exclude norvaline.It is now well established that many aaRSs are incapable of efficient discrimination between cognate and structurally similar non-cognate amino acids in the synthetic reactions. 16,17These enzymes therefore evolved editing mechanisms to hydrolyze non-cognate intermediates (pre-transfer editing) and/or misacylated tRNAs (post-transfer editing). 16ork in our and other laboratories has shown that E. coli LeuRS indeed treats norvaline as a reasonably good substrate in the synthetic reactions; k cat /K m in activation is decreased only 100-fold as compared with leucine, 18,19 while the rate of aminoacyl transfer to tRNA is identical. 19Despite this, we did not observe significant steady-state accumulation of Nva-tRNA Leu in vitro due to the rapid clearance of Nva-tRNA Leu by the post-transfer editing activity 19 located on the separate editing domain known as the CP1 (connective peptide 1) domain. 20,21We also demonstrated that dissociation of Nva-tRNA Leu followed by rebinding and subsequent hydrolysis is a competent kinetic pathway. 19To test if any Nva-tRNA Leu that evades hydrolytic correction is a substrate for ribosomal protein synthesis, its interaction with E. coli EF-Tu:GTP was studied at low (4 °C) and physiological (37 °C) temperatures.Using slightly modified versions of the ribonuclease (RNase) protection 3,4,22 and non-enzymatic hydrolysis protection as-says, 23,24 we show that E. coli EF-Tu does not differentiate between Leu-tRNA Leu and Nva-tRNA Leu at either temperature.The lack of discrimination against norvaline by EF-Tu highlights the importance of rapid hydrolytic correction by LeuRS, demonstrating that it provides the main line of defense against misincorporation of norvaline into proteins.Enhanced understanding of the molecular events that maintain selectivity against non-proteinogenic and/or non-natural amino acids may advance the engineering of proteins with desired features. 25,26

Overexpression and Purification of TEV Protease
A plasmid containing the gene for His-tagged TEV protease was a generous gift from EMBL Protein Expression and Purification Core Facility.E. coli Rosetta cells transformed with the pET24-TEV plasmid were grown to OD 600 0.6-0.8 at 37 °C.The culture was cooled to 15 °C before adding 0.5 mmol dm -3 IPTG, and the cells were allowed to grow for 15 more hours at 15 °C.Cell lysis was performed by sonication in buffer containing 50 mmol dm -3 Tris-HCl (pH = 7.5), 300 mmol dm -3 NaCl, glycerol (φ = 10 %), 0.2 % (v / v) NP-40 and 10 mmol dm -3 β-mercaptoethanol.The lysate was cleared by centrifugation and filtration prior to loading on Ni 2+ -NTA resin.Chromatography was performed as described for EF-Tu.Fractions enriched with TEV protease were pooled and concentrated to 5 mg mL -1 (precipitation was observed at higher concentrations).TEV protease was dialyzed against 25 mmol dm -3 Tris-HCl (pH = 7.5), 150 mmol dm -3 NaCl, glycerol (φ = 10 %), and 5 mmol dm -3 β-mercaptoethanol before storage at -80 °C.

Preparation of LeuRS, tRNA Leu and aa-tRNA Leu
Wild-type E. coli LeuRS and the D345A LeuRS variant defective in hydrolysis of aa-tRNA Leu were overexpressed and purified by affinity chromatography on Ni 2+ -NTA resin, as described. 19,27Because LeuRS copurifies with leucyl-adenylate bound in the active site, a second purification step was performed to ensure its removal. 19Removal of leucyl-adenylate from the LeuRS active site is essential for preparative tRNA Leu misacylation.
Leu-[ 32 P]-tRNA Leu used to determine the fraction of active EF-Tu (see below), was prepared in a slightly different manner, since that assay requires use of a higher aa-tRNA concentration.30 µmol dm -3 tRNA Leu and 10 µmol dm -3 D345A LeuRS were mixed with roughly 200 nmol dm -3 [ 32 P]-tRNA Leu in the standard LeuRS aminoacylation buffer.After approximately 45 min at 37 °C, tRNA Leu was recovered by phenol extraction and ethanol precipitation.The pellet was dissolved in 50 mmol dm -3 sodium acetate (pH = 5.0), applied to a P30-column (Micro Bio-Spin) and dialyzed against 15 mmol dm -3 sodium acetate (pH = 5.0) before storage at -20 °C.The final concentration of aa-tRNA Leu was determined as described. 19

EF-Tu:GDP Activation
Because EF-Tu is purified and stored as the GDP-bound form, it is necessary to convert the protein to the EF-Croat.Chem.Acta 86 (2013) 73.

Determination of the Fraction of Active EF-Tu:GTP
Only a small fraction of the GTP-bound form of EF-Tu is able to bind aa-tRNA. 30The fraction of EF-Tu:GTP active in aa-tRNA binding is generally determined by an RNase protection assay 3,4,22 that relies on the ability of EF-Tu:GTP to protect bound aa-tRNA from RNase digestion.The assay was modified in this work to allow TLC separation of digested and non-digested tRNAs.Varying amounts of EF-Tu:GTP (0-12.5 µmol dm -3 , total protein concentration) in the activation buffer were mixed with saturating amounts of Leu-[ 32 P]-tRNA Leu (600 nmol dm -3 ) for 20 min at 4 °C to allow for ternary complex formation.3 µL of 10 mg mL -1 RNase A were then added to the 30 µL reaction mixture to digest free (unbound) aa-tRNA. 2 µL of reaction mixture were taken at several time points, and were quenched in 4 µL of 1.5 mol dm -3 formic acid to inactivate RNase A. 2-3 µL of this mixture were then spotted onto polyethyleneimine-cellulose plates (Fluka) prewashed in water.Separation of digested from protected aa-[ 32 P]-tRNA was performed by TLC in 0.1 mol dm -3 ammonium acetate and acetic acid (φ = 5 %), followed by quantitation by phosphorimaging.The percentage of aa-[ 32 P]-tRNA protected from RNase A as a function of time was fit to a single exponential equation, and the fraction of aa-[ 32 P]-tRNA initially bound to EF-Tu:GTP was determined from extrapolation to t = 0 (time of RNase A addition).The aa-tRNA fraction (bound at t = 0) was plotted against the total EF-Tu concentration, and the fraction of EF-Tu:GTP molecules capable of binding aa-tRNA was determined from the slope of the linear portion of the plot. 30About 10-15 % of total activated EF-Tu was found to be active in aa-tRNA binding.Throughout this paper, concentrations of EF-Tu:GTP refer to the concentrations of protein capable of aa-tRNA binding, unless otherwise stated.
Control experiments in the absence of EF-Tu were performed to correct for the free aa-tRNA that was not digested within 15 s (first time point) after RNase A addition.Typically, more than 95 % of aa-tRNA was immediately digested and the percentage did not change over time.The remaining aa-tRNA background was subtracted from all experimental data.Control reactions that were performed with varying concentrations of EF-Tu:GDP resulted in immediate digestion of more than 95 % aa-tRNA and matched the reactions performed in the absence of EF-Tu.

Determination of Equilibrium Dissociation Constants, K D at 4 °C
Ternary complex formation was monitored by the modified RNase protection assay as described above.Briefly, subsaturating (1-5 nmol dm -3 ) amounts of aa-[ 32 P]-tRNA were mixed with EF-Tu:GTP and preincubated for 20 min at 4 °C before addition of RNase A. Concentrations of active EF-Tu:GTP were varied in a broad range (5-1400 nmol dm -3 ) to accurately determine K D .The fraction of protected aa-[ 32 P]-tRNA was plotted against the concentration of active EF-Tu:GTP and the data were fit to the hyperbolic equation y = Y 0 × [EF-Tu:GTP] / (K D + [EF-Tu:GTP]) where Y 0 is the maximal protected fraction and K D is the dissociation constant.

Determination of Dissociation Rate Constants, k off at 4 °C
The EF-Tu:GTP:aa-tRNA complex was formed by mixing 600 nmol dm -3 aa-[ 32 P]-tRNA and approximately 1.5 µmol dm -3 active EF-Tu:GTP in EF-Tu activation buffer.The stability of the complex was monitored by the modified RNase protection assay.RNase A was added after a 20 min equilibration period at 4 °C, and time points were collected in a range from 0.15-15 min by mixing 2 µL of reaction mixture with 4 µL of 1.5 mol dm -3 formic acid, followed by TLC analysis as described above.The fraction of aa-[ 32 P]-tRNA protected from RNase A was fit to the single exponential equation y = Y 0 + A × e -k off × t where Y 0 is the y intercept, A is the amplitude, k off is the observed dissociation rate constant and t is time.

Determination of Equilibrium Dissociation Constants, K D at 37 °C
The assay is based on measuring the protective effect of a EF-Tu:GTP:aa-tRNA ternary complex on the nonenzymatic deacylation of aa-tRNA. 23,24The reactions were performed by incubating EF-Tu:GTP with aa-[ 32 P]-tRNA Leu at 37 °C in the activation buffer.aa-[ 32 P]-tRNA Leu was present at 5-10 nmol dm -3 concentration and the concentration of EF-Tu:GTP was varied over a wide range (30-1700 nmol dm -3 ) to most accurately determine K D .Reactions were stopped at different time points by mixing 2 µL aliquots of reaction mixture with 4 µL of quench solution containing 0.75 mol dm -3 sodium acetate (pH = 4.5) and 1.5 g dm -3 SDS.The fraction of aa-tRNA in each time point was determined through P1 nuclease digestion and analysis on TLC plates. 19ata were fit to the single exponential equation y = A × e -k obs × t where A is the amplitude, k obs is the observed non-enzymatic deacylation rate constant, and t is time.Non-enzymatic deacylation rate constants were plotted against concentration of the active EF-Tu:GTP and fit to the equation k obs = k unprotected / (1 + [EF-Tu:GTP]/K D ), where k unprotected is the observed constant for nonenzymatic deacylation rate of aa-tRNA in the absence of EF-Tu:GTP and K D represents the dissociation constant of EF-Tu:GTP:aa-tRNA ternary complex. 24The specificity of the interaction was verified by control reactions performed with several concentrations of EF-Tu:GDP, where the presence of inactive EF-Tu had no protective effect on non-enzymatic deacylation.

Preparation of EF-Tu Suitable for Use in Experiments With Nva-tRNA Leu
3][24] For both assays, high sensitivity to even small contamination by endogenous LeuRS was expected, because (i) low levels of aa-[ 32 P]-tRNA were employed (because of the much higher sensitivity as compared with [ 14 C]-aa-tRNA) and (ii) Nva-tRNA Leu , an efficient (natural) substrate for hydrolytic clearance by LeuRS, 19 was used.Further, EF-Tu:aa-tRNA interactions are generally studied using high EF-Tu (total protein) concentrations, because only a small fraction of EF-Tu:GTP molecules are active in aa-tRNA binding. 30his also makes analysis sensitive to contaminations in the protein sample.To test for the presence of endogenous E. coli LeuRS, the EF-Tu:GDP was tested for Leu-tRNA Leu formation in the standard aminoacylation assay.Significant aminoacylation activity was observed with 12.5 µmol dm -3 EF-Tu:GDP (total protein concentration); comparison with aminoacylation rate achieved by 2 nmol dm -3 LeuRS indicates a contamination level of approximately 0.005 % (Figure 1b).
Next, EF-Tu:GDP was converted to EF-Tu:GTP and tested for interaction with Nva-tRNA Leu using the non-enzymatic hydrolysis protection assay (see below).Significant hydrolysis of Nva-tRNA Leu instead of protection was observed (Figure 1c), confirming that even low levels of copurified LeuRS preclude determination of EF-Tu:Nva-tRNA Leu affinity.To remove these traces of LeuRS, EF-Tu:GDP sample was additionally purified by size-exclusion chromatography (see Experimental section).After this purification, EF-Tu:GDP showed no detectable leucylation activity (Figure 1b) and when activated to EF-Tu:GTP, it efficiently protected Nva-tRNA Leu from non-enzymatic deacylation (Figure 1c).This demonstrates that endogenous LeuRS was completely removed by this additional purification step.

The Modified Ribonuclease Protection Assay
The ribonuclease protection assay 3,4,22 relies on the ability of EF-Tu to protect aa-tRNA from RNase A digestion, thus distinguishing between the bound and free aa-tRNA ligand.The free aa-tRNA is rapidly hydrolyzed by RNase A during a short incubation period, while the bound aa-tRNA remains protected.[ 14 C]-amino acid is generally used to label the aa-tRNA, and digested and protected tRNAs are distinguished by their acidsolubility or acid-insolubility, respectively.Thus, the fraction of aa-tRNA bound to EF-Tu (and thus protected from RNase A digestion) is commonly determined from the radioactivity present in the acid precipitates.Here, we present a modified version of this assay, where the different behavior of digested and protected aa-tRNAs in thin-layer chromatography, instead of different acidsolubilities, are used for separation (Figure 2).The main advantage of this approach is that it is not complicated by the precipitation and filtering steps, and can be easily used in a high-throughput format requiring only a multichannel pipette and 96 well plates.We also used [ 32 P]-tRNA (labeled at the terminal adenosine using tRNA nucleotidyl-transferase) 28,29 to produce aa-[ 32 P]-tRNAs.Use of radiolabeled tRNA was obligatory for studying interactions of EF-Tu with Nva-tRNA Leu because [ 14 C]-Nva is not commercially available.
We first incubated aa-[ 32 P]-tRNA with RNase A, at the same concentrations used in the ribonuclease protection assay, to establish the chromatographic pattern resulting from digestion (Figure 2a).tRNA used in all assays was aminoacylated up to 50-60 % by either leucine or norvaline.Incomplete aminoacylation is not consequential, because the presence of non-aminoacylated tRNA does not influence EF-Tu binding affinity for aa-tRNA. 9Indeed, tRNA samples with less than 30 % of aminoacylated tRNA have previously been successfully used. 9tRNA is rapidly hydrolyzed within 15 s (Figure 2a), confirming that the amount of RNase A is sufficient for rapid and complete digestion.We have also observed a substantial change in chromatographic mobility that allows separate quantitation of digested and non-digested tRNAs.In agreement with previous findings, 30 about 5 % of tRNA remained nondigested or was digested in a way that does not influence its TLC mobility (Figure 2a).This value was subtracted as background from all quantitated data.To perform reliable time-dependent measurements, very rapid inactivation of RNase A digestion is required before TLC analysis.We tested formic acid as a possible quench by preincubating aa-tRNA in 1 mol dm -3 formic acid prior to addition of RNase A. As observed from Figure 2a, 98 % of tRNA was not digested within 30 s or 30 min under these conditions.Thus, RNase A is rapidly inactivated in 1 mol dm -3 formic acid, making it a suitable quench reagent for the RNase A reaction.

EF-Tu:GTP Binds Nva-tRNA Leu and Leu-tRNA Leu With a Similar Affinity
We first used the modified ribonuclease protection assay to test if EF-Tu discriminates between Leu-tRNA Leu and Nva-tRNA Leu .We used this assay to extract both equilibrium (K D ) and rate (k on , k off ) constants describing the interactions between E. coli EF-Tu and either Leu-tRNA Leu or Nva-tRNA Leu , as previously described by Uhlenbeck and colleagues. 4,5,9or equilibrium measurements, various concentrations of EF-Tu:GTP (EF-Tu:GDP was converted to EF-Tu:GTP immediately prior to use) were incubated for 25 min at 4 °C with either Leu-[ 32 P]-tRNA Leu or Nva-[ 32 P]-tRNA Leu .Bound and unbound aa-[ 32 P]-tRNA were distinguished by short incubation with RNase A, where only unbound aa-tRNA has been digested, followed by quenching in formic acid (to inactivate RNase) and separation from the bound aa-tRNA by TLC (for details see Experimental).Digested and protected tRNAs (representing unbound and bound tRNAs, respectively) were independently quantitated (Figure 2b), and the fraction of bound aa-tRNA was calculated and plotted against the concentration of active EF-Tu:GTP (for determination of the fraction of active EF-Tu:GTP see Experimental section) (Figure 3a).Our data show that EF-Tu:GTP binds Leu-tRNA Leu and Nva-tRNA Leu with similar affinity (70 nmol dm -3 and 24 nmol dm -3 , respectively, Figure 3a and Table 1).To the best of our knowledge, these are the first data showing interaction of EF-Tu with norvalylated tRNA.The similar affinities measured for Leu-tRNA Leu and Nva-tRNA Leu are consistent with the data showing incorporation of norvaline in cellular proteins under some conditions. 14,15ext, we determined the dissociation rate constants of Leu-tRNA Leu and Nva-tRNA Leu (k off ) from their corresponding EF-Tu:GTP:aa-tRNA ternary complexes using the modified version of ribonuclease protection assay.The rationale was to examine if kinetics of association (k on and k off ) significantly differs for these two aa-tRNAs, in spite of the similar overall K D .EF-Tu:GTP and aa-tRNA were incubated for 25 min at 4 °C followed by RNase A addition.Time points were taken at regular intervals, and the reaction was quenched in 1 mol dm -3 formic acid.Digested and protected tRNAs were separated as described for the equilibrium measurements (Figure 2).The fraction of remaining bound aa-tRNA was plotted vs. time, and first order rate constant representing k off was extracted (Figure 3b).Similar to our findings with respect to equilibrium constants, the dissociation rate constants (k off ) for Leu-tRNA Leu and Nva-tRNA Leu are also highly similar (Figure 3a and Table 1).Taken together, our data clearly show that neither equilibrium binding nor association kinetics differ significantly between EF-Tu:GTP:Leu-tRNA Leu and EF-Tu:GTP:Nva-tRNA Leu ternary complexes.Thus, E. coli EF-Tu does not distinguish between Leu-tRNA Leu and Nva-tRNA Leu .

Discrimination of Norvaline by EF-Tu:GTP is not Enhanced at the Physiologically Relevant Temperature
The data so far presented describes the EF-Tu:GTP:aa-tRNA interactions at 4 °C, a temperature that is not physiologically highly relevant, but is commonly used in EF-Tu binding studies.Two main advantages of working at lower temperatures are (i) higher affinity of EF-Tu:GTP for aa-tRNA and (ii) significantly slower aa-tRNA dissociation rate allowing manual sampling of time points. 4,31However, there is a possibility that EF-Tu:GTP discriminates better against Nva-tRNA Leu at the physiologically more relevant temperature.To experimentally address this issue, we used a different assay, 23,24 better suited for the work at higher temperatures.This assay is based on protection of aa-tRNA from non-enzymatic hydrolysis in solution, when it is bound to EF-Tu:GTP.The GDP-bound form of EF-Tu was firstly converted to the GTP-bound form prior to mixing with aa-[ 32 P]-tRNA at 37 °C.At certain time points, reaction aliquots were quenched, tRNA was degraded using P1 nuclease, and products were analyzed by TLC as described. 19,27Representative time courses obtained at different concentrations of active EF-Tu:GTP are shown in Figure 4a.As expected, at higher concentrations of EF-Tu:GTP, the pre-equilibrium is shifted toward preferential binding of aa-tRNA, thus decreasing the rate of non-enzymatic hydrolysis (i.e.better protection is observed).As a control, the same experiment was performed with EF-Tu:GDP (Figure 4b).No protection of Leu-tRNA Leu was observed, confirming the specificity of interactions measured in Figure 4a.The dependence of k obs vs. concentration of the active EF-Tu:GTP yields K D (Figure 4c and Table 1).Interestingly, a similar level of discrimination is observed at 37 °C and 4 °C, which further demon-  strates that Nva-tRNA Leu and Leu-tRNA Leu are equally good substrates for EF-Tu:GTP-mediated transport to the ribosome.

DISCUSSION
Quality control of protein translation is manifested at several steps: formation of aa-tRNAs by aaRSs, EF-Tu:GTP dependent delivery of aa-tRNAs to the ribosome, and the subsequent mRNA decoding, where the anticodon of aa-tRNA is matched with the cognate codon to ensure incorporation of the esterified amino acid at the appropriate position in the growing polypeptide chain.Each of these steps possesses an inherent error frequency and mechanism to maintain error rates within the level tolerable in protein synthesis (10 -3 -10 -4 ). 16rompted by the findings that the non-proteinogenic amino acid norvaline partially evades translational proofreading mechanisms, and is thus incorporated into proteins in place of leucine under some conditions in vivo, 14,15 we have recently analyzed in detail the capacity of E. coli LeuRS to discriminate against formation of Nva-tRNA Leu . 19In agreement with previous findings, 18 norvaline was indeed determined to be a reasonably good substrate for the LeuRS synthetic reactions.However, accumulation of Nva-tRNA Leu was not observed due to its rapid hydrolysis (the single turnover rate constant is 300 s -1 ) within the LeuRS CP1 editing site. 19ere, we further explore the capacity of Nva-tRNA Leu to bind EF-Tu:GTP.The rationale was to determine whether any Nva-tRNA Leu that evades LeuRS hydrolytic editing may participate efficiently in the subsequent step in translation.
First, we tested equilibrium binding of Nva-tRNA Leu and Leu-tRNA Leu to EF-Tu:GTP at 4 °C using a modified version of the commonly used ribonuclease protection assay to extract K D values.Previous work has shown that all elongator aa-tRNAs bind E. coli EF-Tu:GTP similarly, with only a 12-fold range in the K D values. 3Thus, the 3-fold difference in K D values for Nva-tRNA Leu and Leu-tRNA Leu (Table 1) strongly suggests that Nva-tRNA Leu is indistinguishable from Leu-tRNA Leu and other elongator cognate aa-tRNAs regarding its interaction with EF-Tu.Moreover, the observed K D values are very similar to the previously determined K D for E. coli Phe-tRNA Phe under comparable ionic strength conditions. 32Analysis of association kinetics revealed the same pattern.k off for Nva-tRNA Leu and Leu-tRNA Leu differs by less than 2-fold (Table 1) and the values are highly similar to the previously determined k off for Phe-tRNA Phe . 31,32In general, EF-Tu K D values are less accurate than k off values because of the error-prone determination of the active EF-Tu:GTP fraction, which is a prerequisite for K D extraction (Figure 3).Because k on was shown to be constant for different aa-tRNAs, 32 K D is often calculated from the k on value 32 (1.1 × 10 5 mol -1 dm 3 s -1 ) and the experimentally determined k off . 5,12,30Here, we calculated k on from the experimentally measured K D and k off values for both Leu-tRNA Leu and Nva-tRNA Leu .The values obtained (Table 1) are very similar to each other and to the previously determined k on value.This provides considerable confidence in the reported thermodynamic and kinetic parameters and strongly supports the conclusion that Nva-tRNA Leu is not discriminated in translation at the level of EF-Tu:GTP binding.
Additional proof was obtained from the analysis of EF-Tu:GTP interactions with both Nva-tRNA Leu and Leu-tRNA Leu at the physiologically more relevant temperature of 37 °C.Here we used an assay that relies on protection of aa-tRNA from solution-based non-enzymatic hydrolysis when bound to EF-Tu:GTP. 23,24Again, the extracted K D values for Nva-tRNA Leu and Leu-tRNA Leu (Table 1) were highly similar, demonstrating that discrimination against norvaline is independent of temperature.We also show that aa-tRNA binds EF-Tu:GTP weaker at higher temperatures, consistent with previous observations. 4,31The absence or presence of the N-terminal His 6 -tag on EF-Tu did not influence its interactions with aa-tRNAs (Table 1).
According to the thermodynamic compensation model, 4 the contributions of the esterified amino acid and the tRNA body are independent of one another, but compensate such that all cognate aa-tRNA pairs have similar binding affinities.Because fine tuning of binding affinities by the compensation mechanism is disturbed in misacylated tRNAs, these species bind EF-Tu:GTP over a broad range of affinities, varying from 60-fold weaker  to 120-fold tighter as compared with cognate aa-tRNA pairs. 5][12] Structural 33,34 and thermodynamic analyses 5 revealed that amino acids bind in the same pocket on the surface of EF-Tu, but establish slightly different contacts that result in different binding affinities.Lack of discrimination against Nva-tRNA Leu suggests that both norvaline and leucine establish interactions with EF-Tu:GTP that are thermodynamically comparable.Inspection of Thermus thermophilus EF-Tu interactions with specifically designed misacylated tRNAs revealed that valine binds approximately 2-fold weaker than the slightly bigger isoleucine. 5Comparison of the measured k off values (Table 1) revealed that norvaline, although smaller, binds EF-Tu approximately 1.5-fold tighter than leucine.Thus, it is likely that norvaline compensates for the lack of the methyl group binding energy by establishing better interactions of its unbranched side chain within the EF-Tu binding pocket.
Our extensive kinetic analysis demonstrated that wildtype LeuRS very efficiently clears Nva-tRNA Leu in vitro. 19However, anything that disturbs the kinetic partitioning of Nva-tRNA Leu between post-transfer editing hydrolysis and dissociation (either via compromising hydrolytic activity or stimulating dissociation) may result in accumulation of Nva-tRNA Leu .It is not yet understood what factors may influence kinetic partitioning of Nva-tRNA Leu in vivo.However, what we clearly established here is that after Nva-tRNA Leu is released from LeuRS, it binds EF-Tu as efficiently as Leu-tRNA Leu or any other elongator aa-tRNA.This explains the occurrence of norvaline incorporation in place of leucine in vivo, under conditions where its accumulation allows efficient activation by LeuRS. 14,15aken together, these findings strongly suggest that hydrolytic editing by LeuRS serves as the main quality control checkpoint against incorporation of norvaline into cellular proteins.Indeed, when LeuRS hydrolytic editing was compromised, a significant substitution of leucine by norvaline was observed in a reporter protein in vivo. 18Similarly, mistranslation of phenylalanine codons by tyrosine parallels the utilization of an editing-deficient phenylalanyl-tRNA synthetase (PheRS) in poly(U)-directed polyTyr/polyPhe synthesis assay.In this case it was shown that mistranslation occurs in part from the inability of E. coli EF-Tu to discriminate between Phe-tRNA Phe and Tyr-tRNA Phe . 31One may argue that because of efficient editing by aaRSs, such as PheRS 35,36 and LeuRS, 19,37,38 EF-Tu was not subject to evolutionary pressure to develop stringent discrimination against Tyr-tRNA Phe and Nva-tRNA Leu .11]24 The failure of EF-Tu to discriminate among some aminoacyl-tRNA substrates places the main burden of translational fidelity on the corresponding aaRSs.It appears that the interplay between attainable accuracies in cognate aa-tRNA formation and EF-Tu recognition may have driven evolution, at least in the case of LeuRS, towards acquisition of the highly efficient hydrolytic site that prevents accumulation of Nva-tRNA Leu for mistranslation.

Figure 2 .
Figure 2. Characteristic RNase digestion pattern obtained by thin-layer chromatography.The thin-layer chromatogram represents a time course obtained by incubating Leu-[ 32 P]-tRNA Leu with RNase A in the absence of EF-Tu:GTP (left side of the panel, -EF-Tu:GTP) and a quench control performed by adding RNase A to Leu-[ 32 P]-tRNA Leu mixed with 1 mol dm -3 formic acid (right side of the panel, quench control).Digested and non-digested tRNAs were separately quantitated with ImageQuant software and the fraction of non-digested tRNA was calculated by dividing the intensity of non-digested tRNA with the total intensity.To calculate the fraction of non-digested aa-tRNA, the fraction of non-digested tRNA was divided by the fraction of aa-tRNA initially present in the sample (a).Representative thin-layer chromatogram of a time course obtained in the modified RNase protection assay where RNase A was added to a mixture of Leu-[ 32 P]-tRNA Leu and EF-Tu:GTP preincubated at 4 °C (b).

Table 1 .
Thermodynamic and kinetic parameters describing EF-Tu:GTP:aa-tRNA interactions.The values represent the best fit value ± s.e.m. of three independent experiments.