Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

Abstract

To better understand why aminoacyl-tRNAs (aa-tRNAs) have evolved to bind bacterial elongation factor Tu (EF-Tu) with uniform affinities, mutant tRNAs with differing affinities for EF-Tu were assayed for decoding on Escherichia coli ribosomes. At saturating EF-Tu concentrations, weaker-binding aa-tRNAs decode their cognate codons similarly to wild-type tRNAs. However, tighter-binding aa-tRNAs show reduced rates of peptide bond formation due to slow release from EF-Tu•GDP. Thus, the affinities of aa-tRNAs for EF-Tu are constrained to be uniform by their need to bind tightly enough to form the ternary complex but weakly enough to release from EF-Tu during decoding. Consistent with available crystal structures, the identity of the esterified amino acid and three base pairs in the T stem of tRNA combine to define the affinity of each aa-tRNA for EF-Tu, both off and on the ribosome.

The ternary complex of bacterial elongation factor Tu (EF-Tu), GTP and aminoacyl-tRNA (aa-tRNA) binds to the ribosome and participates in a multistep decoding pathway in which GTP is hydrolyzed, EF-Tu•GDP is released, and the aa-tRNA enters the ribosomal A site (1–6). Although all elongator aa-tRNAs bind EF-Tu•GTP with similar affinities (7–9), studies with misacylated tRNAs reveal that the protein shows substantial specificity for both the esterified amino acid and the tRNA body (10–12). The nearly uniform EF-Tu binding affinity observed for tRNAs acylated with their correct (cognate) amino acid occurs because the sequence of each tRNA has evolved to compensate for the variable thermodynamic contribution of the esterified amino acid. Thus, weak-binding esterified amino acids such as glycine and alanine have corresponding tRNAs that bind the protein tightly, while tight-binding amino acids such as tyrosine or glutamine have corresponding tRNAs that bind poorly. The crystal structure of Thermus aquaticus EF-Tu•GTP bound to Saccharomyces cerevisiae Phe-tRNA^Phe (13) reveals that the protein primarily forms extensive interactions with the helical phosphodiester backbone of the acceptor and T stems of tRNA^Phe. Recent protein (14) and tRNA (15, 16) mutagenesis experiments indicate that much of the specificity is the result of interactions made between three amino acids of EF-Tu and three adjacent base pairs in the T stem (16). Additional mutagenesis experiments indicate that the thermodynamic contribution of each of the three base pairs is independent of the others, making it possible to adjust the affinity of aa-tRNAs to EF-Tu in a predictable manner.

Although a detailed structural and thermodynamic understanding of how EF-Tu achieves uniform binding with different aa-tRNAs is beginning to emerge, the underlying selective pressures that lead to uniform binding are less clear. While aa-tRNAs must bind EF-Tu tightly enough to participate in translation, the high intracellular concentration of EF-Tu (17) ensures that they do not significantly compete with one another for the protein. It therefore seems unlikely that a minimum threshold binding affinity would provide sufficient selective pressure to ensure the observed uniform binding properties. Additional selective pressure could potentially come at the step in translation where aa-tRNAs are released from EF-Tu•GDP prior to peptide bond formation. This could limit the affinity to a maximal threshold, thereby selecting for uniform binding. The recent crystal structure of a ribosome-bound ternary complex trapped just before release of the aa-tRNA from EF-Tu (18) reveals that although the ribosome forms multiple contacts with both the tRNA and EF-Tu, the overall arrangement of the three domains of EF-Tu and its interface with aa-tRNA is quite similar to that of the free ternary complex. This suggests that the thermodynamic interplay between the esterified amino acid and the T-stem sequence may also occur during decoding and provide an additional selective pressure for uniform binding. This paper tests this possibility by examining the decoding properties of aa-tRNAs that have been altered to have either an increased or decreased affinity for EF-Tu by changing the T-stem sequence or introducing a different esterified amino acid.

Results

Derivatives of Escherichia coli with Differing Affinities to E. coli EF-Tu.

The E. coli species that decodes GUC/U codons was chosen because its tRNA body has an intermediate affinity for EF-Tu (10), making it possible to both increase and decrease its binding affinity by mutating the T stem. In addition, this tRNA is one of the few in E. coli that lacks posttranscriptional modifications in its anticodon hairpin, thereby maximizing the likelihood that it will decode effectively as an unmodified tRNA. Based upon the binding affinities of individual base-pair substitutions in S. cerevisiae tRNA^Phe to T. thermophilus EF-Tu (16), T-stem mutations of (Fig. 1A) were designed to bind tighter (T1 to T3), weaker (W1 to W3), or similar (Ψ) to the wild-type (WT) tRNA sequence. After aminoacylation with [³H]-valine, a ribonuclease protection assay was used to determine the dissociation rates of WT and the seven mutants of tRNA^Val from E. coli EF-Tu•GTP at 4 °C (Fig. 1B, Table 1) (19, 20). Dissociation rates for several tRNAs were also determined at 20 °C, and k_off values were ninefold to 15-fold faster than at 4 °C (Table S1). As previously observed with other tRNAs,(12, 21, 22) the unmodified WT bound EF-Tu very similarly to native, fully modified consistent with the absence of modifications in the EF-Tu binding site. The seven mutations showed the desired broad range of binding affinity to E. coli EF-Tu that is similar to the range in affinities observed for different tRNA bodies with T. thermophilus EF-Tu (10, 16). T1 to T3 bound from 0.5 to 1.0 kcal/mol tighter, W1 to W3 bound from 1.0 to 2.0 kcal/mol weaker, and Ψ bound nearly the same as the WT tRNA^Val. These measured values correlate closely with the values predicted by adding the ΔΔG° values obtained from data measuring the binding of single base-pair substitutions in S. cerevisiae tRNA^Phe to T. thermophilus EF-Tu (Fig. 1C). This not only supports the notion that the thermodynamic contributions of the three T-stem base pairs are independent but indicates that both E. coli and T. thermophilus EF-Tu interact with aa-tRNAs in a thermodynamically similar manner.

Fig. 1.

Engineering Val-tRNA^Val mutants with altered EF-Tu binding affinities. (A) E. coli with mutated T-stem base pairs in box. T-stem sequences of WT and seven mutant tRNA transcripts are on the right. (B) Individual rates of dissociation (k_off) for WT ● (12 × 10^-4 s^-1), W1 ♦ (170 × 10^-4 s^-1), T1 ▪ (2.0 × 10^-4 s^-1), and native ▴ (5.5 × 10^-4 s^-1) Val-tRNA^Val from E. coli EF-Tu at 0 °C in RB buffer using the ribonuclease protection assay. Means and standard errors from multiple determinations are in Table 1. (C) Comparison of the free energies of binding (ΔG°) between Val-tRNA^Val T-stem mutants to E. Coli EF-Tu with measured ▪ or calculated ♦ free energies of the same mutants in S. cerevisiae Phe-tRNA^Phe binding to T. thermophilus EF-Tu. The line corresponds to a least square fit with a slope of 1.1.

Table 1.

k_off and k_pep values of Val-tRNA^Val mutants

tRNA	Rate of aa-tRNA dissociation from EF-Tu 104 × koff(s-1)	Free energy of aa-tRNA Binding to EF-Tu ΔG° (kcal/mol)	Rate of peptide bond formation kpep(s-1)
Native	5.6 (±) 0.4	−10.4 (±) 0.1	3.3 (±) 0.4
WT	13 (±) 4.0	−9.9 (±) 0.3	4.5 (±) 0.7
T1	2.0 (±) 0.5	−10.9 (±) 0.3	0.32 (±) 0.02
T2	3.4 (±) 1.3	−10.6 (±) 0.2	0.55 (±) 0.04
T3	5.3 (±) 0.5	−10.4 (±) 0.1	1.4 (±) 0.2
Ψ	22 (±) 5.0	−9.6 (±) 0.2	4.5 (±) 0.8
W1	110 (±) 6.7	−8.8 (±) 0.1	5.4 (±) 2.3
W2	130 (±) 39	−8.6 (±) 0.3	6.0 (±) 2.5
W3*	950 (±) 210	−7.6 (±) 0.2	3.3 (±) 0.1

Standard errors are calculated from at least three independent determinations.

^*k_off value extrapolated from lower NH₄Cl concentrations.

Decoding Properties of tRNA^Val T-stem Mutants.

Three quantitative assays were used to evaluate the ability of ternary complexes made with native tRNA^Val and the unmodified WT, T1, and W1 tRNAs to decode the GUC codon on E. coli ribosomes containing fMet-tRNA^fMet in the P site (Fig. 2, Table 2). The experiments were performed at 20 °C in a buffer containing 10 mM MgCl₂ to ensure that the unmodified tRNAs were fully folded (23–25). The first assay measures the equilibrium binding constant of the ternary complex to ribosomes by using the active site mutant of EF-Tu(H84A) to prevent GTP hydrolysis (26, 27). The resulting equilibrium constant reflects the initial binding, codon recognition, and GTPase activation steps of the decoding pathway (28). As shown in Fig. 3A, all of the tRNA^Val derivatives show very similar K_d values. Thus, neither the tRNA modifications nor the T-stem mutations significantly alter the initial interaction of the ternary complex with the ribosome.

Fig. 2.

Steps of EF-Tu catalyzed selection of aa-tRNA by the ribosome This simplified scheme, adapted from ref. 28, indicates the steps that were assayed to evaluate the activities of the mutants of Val-tRNA^Val on E. coli ribosomes.

Table 2.

Performance of Val-tRNA^Val mutants in decoding

tRNA	A/T site affinity Kd (nM)	Maximal rate of GTP hydrolysis kGTPmax (s-1)	Half maximal [70S] for GTP hydrolysis K1/2 (μM)
Native	1.1 (±) 0.3	27 (±) 9.7	2.6 (±) 1.9
WT	0.8 (±) 0.2	27 (±) 7.4	2.7 (±) 1.4
T1	0.5 (±) 0.2	41 (±) 13	2.2 (±) 1.4
W1	2.1 (±) 0.7	28 (±) 8.3	1.6 (±) 1.2

Standard errors for A/T site K_d and k_pep are calculated from at least three independent determinations.

Standard errors for k_GTPmax and K_1/2 are fit to the Michaelis–Menton equation with at least six k_GTPapparent determinations per curve.

Fig. 3.

Activities of Val-tRNA^Val mutants in decoding. Individual determinations of the performance of WT ●, W1 ♦, T1 ▪, and native ▴ Val-tRNA^Val on encoded ribosomes at 20 °C in RB using three assays. Means and standard errors of multiple determinations are in Table 2. (A) Binding affinities of H84A (GTPase deficient) EF-Tu ternary complexes to the ribosomal A/T site using < 0.23 nM ternary complex. Lines are best fit simple binding curves with K_d = 0.8 nM, 1.5 nM, 0.7 nM, and 1.4 nM, respectively. (B) Rates of γ-[³²P]-GTP hydrolysis (k_GTP) at 200 nM ternary complex and 2.5 μM ribosomes. Lines are best fit single exponential rates with k_GTP = 10 s^-1, 16 s^-1, 7.5 s^-1, 11 s^-1, respectively. (C) Ribosome saturation curve fit to the Michaelis–Menten equation for K_1/2 and k_GTPmax(see Table 2). (D) Rates of peptide bond formation (k_pep) measured at 25 nM ternary complex and 1 μM ribosomes. Lines are single exponential fits with k_pep = 5.7 s^-1, 8.1 s^-1, 0.34 s^-1, and 3.8 s^-1, respectively.

The second assay uses ternary complexes made with γ-[³²P]-GTP to measure the rate of GTP hydrolysis as a function of encoded ribosome concentration. This rate reflects a conformational change in EF-Tu that occurs before bond cleavage (28–30). As shown in Fig. 3B, the rate of GTP hydrolysis at 2.5 μM ribosomes differed by about twofold between the four Val-tRNA^Val derivatives tested. When the rates of GTP hydrolysis were plotted as a function of ribosome concentration (Fig. 3C), values of K_1/2 and k_GTPmax (Table 2) also differ by about twofold. Thus, either increasing (T1) or decreasing (W1) the affinity of Val-tRNA^Val for EF-Tu by about 1.0 kcal/mol had little effect on the ability of the ternary complex to catalyze GTP hydrolysis on the ribosome. The tRNA modifications also had little effect on k_GTP.

The final assay measures k_pep, the rate of ribosome-catalyzed formation of fMetVal-tRNA^Val. This final step in decoding encompasses the release of both inorganic phosphate and Val-tRNA^Val from EF-Tu•GDP, the accommodation of the Val-tRNA^Val into the A site and the formation of the peptide bond. It remains uncertain which one of these substeps limits the value of k_pep for wild-type aa-tRNAs, and it may depend upon the assay conditions and the aa-tRNA that are used (28, 31, 32). Here k_pep is measured with ternary complexes containing 3′-[³²P]-labeled Val-tRNA^Val using a P1 nuclease assay (33). The ternary complexes containing native tRNA^Val, the WT, and W1 derivatives all show similar k_pep values and extents of reaction (Fig. 3D, Table 2). Strikingly, the ternary complex containing tight-binding T1 tRNA^Val is fully active but shows a 14-fold slower k_pep value than WT. This slow k_pep can potentially be explained by its slow release from EF-Tu•GDP (Fig. 2). To further test this, k_pep values for the remaining five tRNA^Val derivatives were determined (Table 1). The values of k_pep for both the weaker-binding W2 mutation and the very weak-binding W3 mutation are similar to WT, although higher concentrations of EF-Tu are required in the latter case to achieve high levels of dipeptide. The Ψ mutation, which binds similarly to WT, also shows a similar k_pep value. In contrast, T2 and T3, which both bind to EF-Tu more tightly, are fully active but show significantly slower k_pep values. Taken together, the data indicate that while removing the modifications or weakening the affinity to EF-Tu has little effect on the rate of peptide bond formation, tightening the interaction with EF-Tu clearly reduces it.

Rescuing the Slow k_pep with a Sequence-Specific EF-Tu Mutation.

If the reduced k_pep values observed with the three hyperstabilized tRNA^Val derivatives are the result of slow release from EF-Tu•GDP, it should be possible to reverse this effect with an EF-Tu mutation that destabilizes tRNA binding. To test this, we made use of a contact between EF-Tu and tRNA that stabilizes the interaction in a sequence-specific manner (15). In the ternary complex structure of E. coli EF-Tu with E. coli Phe-tRNA^Phe (PDB ID code 1OB2), E378 makes a hydrogen bond with the amino group of G63 (Fig. 4A). Mutation of the homologous E390 in T. thermophilus EF-Tu to an alanine reduced the binding affinity of the protein to tRNAs containing a G at either position 51 or 63 but not to tRNAs lacking a G (15). A similar hydrogen bond is observed between E390 of T. thermophilus EF-Tu and G63 of E. coli Thr-tRNA^Thr in the structure of the ternary complex bound to T. thermophilus ribosomes in the presence of kirromycin (Fig. 4B) (18). Because this structure depicts EF-Tu after GTP hydrolysis, the hydrogen bond could affect the rate of release of aa-tRNA prior to accommodation. A more recent structure of the ribosome-bound ternary complex before GTP hydrolysis is very similar (34).

Fig. 4.

The EF-Tu•tRNA interface maintains similar interactions on the ribosome. (A) Cocrystal structure of E. coli EF-Tu bound to S. cerevisiae Phe-tRNA^Phe with kirromycin (PDB) highlighting the interactions made between E378, Q329, and T338 10B2 (E390, Q341, T350 in T. thermophilus) with base pair 51–63 and the 2′ OH of bases 64 and 65. (B) Cocrystal structure of T. thermophilus EF-Tu bound to E. coli Thr-tRNA^Thr stalled on the ribosome with kirromycin post GTP hydrolysis (PDB ID code 2WRN) (18).

To confirm that the E378A mutation of E. coli EF-Tu shows the expected specificity for binding to the tRNA^Val derivatives, k_off values were determined for ternary complexes of this mutant protein with WT, Ψ, and the three tight tRNA^Val derivatives (Table 3). The k_off for the WT Val-tRNA^Val was not significantly affected by the E378A mutation, consistent with the fact that it contains a U51-A63 base pair. However, the Ψ, T1, and T2 derivatives, which contain G51-C63, dissociate fivefold to sixfold faster from EF-Tu(E378A) than they do from WT EF-Tu (Table 1), consistent with the presence of a stabilizing hydrogen bond with G51. In contrast, the tight-binding derivative T3, which contains an A51-C63 pair, is not affected by the E378A mutation. While it is unclear how the A51-C63 mismatch stabilizes EF-Tu binding, it appears to do so without the use of E378 and therefore is a valuable control.

Table 3.

k_off and k_pep values of Val-tRNA^Val mutants with EF-Tu(E378A)

tRNA	Rate of aa-tRNA dissociation from EF-Tu 104 × koff (s-1)	Free energy of aa-tRNA binding to EF-Tu ΔG° (kcal/mol)	Rate of peptide bond formation kpep (s-1)
WT	18 (±) 8.9	−9.7 (±) 0.5	4.4 (±) 1.5
T1	10 (±) 3.7	−10.0 (±) 0.4	2.7 (±) 0.4
T2	19 (±) 6.7	−9.7 (±) 0.4	3.1 (±) 0.3
T3	5.2 (±) 0.4	−10.4 (±) 0.1	0.59 (±) 0.3
Ψ	140 (±) 36	−8.6 (±) 0.3	3.7 (±) 0.6

Standard errors are calculated from at least three independent determinations.

k_pep values for WT, Ψ, T1, T2, and T3 versions of Val-tRNA^Val were then determined using ternary complexes containing EF-Tu(E378A) (Table 3). The value of k_pep for the WT tRNA with the mutant EF-Tu (4.4 s^-1) was identical to that observed using WT EF-Tu (4.5 s^-1). Because WT Val-tRNA^Val has a U51-A63 base pair and is not stabilized by the E378 residue, this establishes that EF-Tu(E378A) is fully active in decoding. However, the k_pep values for the tightly binding T1 and T2 tRNAs with EF-Tu(E378A) are 2.7 s^-1 and 3.3 s^-1, respectively. Because these values are substantially faster than the 0.32 s^-1 and 0.55 s^-1 observed with WT EF-Tu, it is clear that the EF-Tu mutation that weakens binding to the hyperstabilized T1 and T2 tRNA^Val derivatives also reverts their abnormally slow k_pep values. This experiment shows that the hydrogen bond between E378 and G63 in the ribosome-bound ternary complex contributes to its stability and thereby influences the rate of release of an aa-tRNA into the A site.

Two critical control experiments are the k_pep measurements of the Ψ and T3 tRNAs with EF-Tu(E378A). Despite the fact that Ψ contains a G51-C63 and thus binds more weakly to the E378A protein, k_pep for Ψ with EF-Tu(E378A) (3.7 s^-1) is similar to WT EF-Tu (4.5 s^-1). This experiment shows that the E378A mutation only stimulates k_pep when the tRNA is hyperstabilized for EF-Tu binding. The second control shows that the slow k_pep observed for the T3 tRNA^Val with WT EF-Tu (1.4 s^-1) remains slow with EF-Tu(E378A) (0.6 s^-1). Because the A51-C63 pair present in T3 is not stabilized by E378, its k_pep is unaffected by the E378A mutation. This control shows that when the ternary complex is hyperstabilized in a different way, the slow k_pep no longer requires the stabilizing contact using E378. Taken together, the experiments with EF-Tu(E378A) confirm that the slow k_pep values observed for the hyperstabilized tRNA^Val derivatives are caused by the slow release from EF-Tu.

Contribution of the Esterified Amino Acid to k_pep.

While the above experiments establish that the sequence of the T stem can influence the rate of release from EF-Tu•GDP during decoding, it is not clear whether the identity of the esterified amino acid can influence this step in a manner similar to what is observed in the formation of the ternary complex. In the ribosome-bound ternary complex (18), the position of the esterified amino acid is different than in the free ternary complex, so it is uncertain whether the thermodynamic effect of the amino acid would be similar. One way to detect an effect of the amino acid on k_pep is to introduce the hyperstabilizing T1 sequence into other E. coli tRNAs. Because the specificity of EF-Tu for tRNA is primarily defined by the T-stem sequence (16), the T1 chimeras are expected to have a similar tight affinity to EF-Tu•GTP, and any difference in k_off should primarily reflect differences in the esterified amino acid. This T1 “upgrade” mutation was introduced into the bodies of E. coli , , , and , and the resulting chimeras were acylated with their corresponding cognate amino acids. k_off values for the chimeras were compared with the WT aa-tRNAs in Table 4. In general, the T1 mutation stabilized the binding of each aa-tRNA to EF-Tu•GTP in the expected manner. Because and are tight-binding tRNA sequences (10), their T1 chimeras only bound EF-Tu twofold and fourfold tighter than their WT counterparts. The T1 chimera of , an intermediate-binding tRNA, binds 56-fold tighter than the WT tRNA^Phe. For the T1 chimera of the very weak-binding , dissociation from EF-Tu GTP was too slow to obtain a reliable k_off, even when the ionic strength was increased to 3 M NH₄Cl or the temperature raised to 20 °C. An estimated k_off for the T1 Tyr-tRNA^Tyr, calculated based on its sequence (16), was 62-fold tighter than WT tRNA^Tyr.

Table 4.

k_off and k_pep values of WT and T1 mutations

tRNA	Rate of aa-tRNA dissociation from EF-Tu 104 × koff (s-1)	Free energy of aa-tRNA binding to EF-Tu ΔG° (kcal/mol)	Rate of peptide bond formation kpep (s-1)
Ala-tRNAAla WT	21 (±) 2.3	−9.7 (±) 0.1	6.8 (±) 0.6
Ala-tRNAAla T1	4.8 (±) 0.7	−10.5 (±) 0.2	2.6 (±) 0.1
Gly-tRNAGly WT	29 (±) 1.7	−9.5 (±) 0.1	1.4 (±) 0.6
Gly-tRNAGly T1	17 (±) 1.3	−9.8 (±) 0.1	0.81 (±) 0.3
Phe-tRNAPhe WT	35 (±) 7.6	−9.4 (±) 0.1	2.3 (±) 1.1
Phe-tRNAPhe T1	0.6* (±) 0.1	−11.6 (±) 0.2	0.18 (±) 0.11
Tyr-tRNATyr WT	9.9 (±) 0.1	−10.1 (±) 0.1	1.4 (±) 0.5
Tyr-tRNATyr T1	0.16†	−12.3	0.025 (±) 0.012

^*Beyond limit of assay, estimated value.

^†k_off value extrapolated from higher NH₄Cl concentrations.

Standard errors are calculated from at least three independent determinations.

k_pep values for the eight tRNAs are also shown in Table 4. The four unmodified WT aa-tRNAs had k_pep values that were reasonably similar to WT tRNA^Val (4.5 s^-1). The slightly slower rates for (1.4s^-1) and (2.3 s^-1) probably reflect the absence of modifications in the anticodon hairpin, which is known to weaken ribosome binding (35). The slightly slower rate for (1.4 s^-1) probably reflects the lower pKa of glycine (36). The k_pep values for the four T1 derivatives were all slower than the corresponding WT tRNAs and correlated closely with their k_off rates. Thus, k_pep values for the T1 derivatives of and were only twofold to threefold slower than the WT tRNAs. However, k_pep of the T1 mutant of was 13-fold slower than that of WT Phe-tRNA^Phe. Finally, the T1 mutation of Tyr-tRNA^Tyr shows a very slow k_pep of 0.025 s^-1, which is 57-fold slower than measured for WT Tyr-tRNA^Tyr. The 104-fold range in k_pep for the T1 mutations correlates remarkably well with the greater than 100-fold difference in their k_off values, supporting the view that the compensatory relationship between the esterified amino acid and tRNA body observed for the free ternary complex occurs in an identical way during decoding.

Although the above data strongly suggests that the identity of the esterified amino acid can contribute to the rate of release from EF-Tu•GDP during decoding, experiments with misacylated-tRNAs are needed to show this directly. We made use of the observation that tRNA^Val can be misacylated by high concentrations of yeast-PheRS, and the resulting Phe-tRNA^Val binds EF-Tu twofold to threefold tighter than Val-tRNA^Val (10). Table 5 shows that the k_pep values of WT and T1 are 4.3- and 2.2-fold slower when phenylalanylated than when valylated, which agrees reasonably well with the twofold to threefold difference reported in k_off(10). In contrast, the k_pep of the phenylalanylated W1 tRNA (5.1 s^-1) is the same as when it is valylated (5.4 s^-1) because the relatively large (1.1 kcal/mol) destabilizing effect of the W1 mutation more than offsets the relatively small (0.5 kcal/mol) effect of introducing the phenylalanine, so the aa-tRNA is not hyperstabilized. In order to show that misacylation can also lead to a faster k_pep, the acceptor stem of the T1 derivative of tRNA^Tyr was mutated to contain a G3-U70 base pair so it could be alanylated by AlaRS(37). As shown in Table 5, k_pep for this Ala-tRNA^Tyr is 61-fold faster than when esterified with the tight-binding tyrosine. These experiments with misacylated tRNAs confirm that the esterified amino acid contributes to the rate of release from EF-Tu•GDP during decoding in a compensatory manner that is very similar to that observed for the formation of ternary complex.

Table 5.

k_pep of misacylated tRNAs

tRNA	Rate of peptide bond formation kpep (s-1)
Phe-tRNAVal WT	1.5 (±) 0.7
Phe-tRNAVal T1	0.16 (±) 0.04
Phe-tRNAVal W1	5.1 (±) 0.8
Ala-tRNATyr T1	1.4 (±) 0.4

Standard errors are calculated from at least three independent determinations.

Discussion

Several conclusions of this paper can be conveniently summarized by plotting k_off, the rate of dissociation of aa-tRNA from EF-Tu•GTP, versus k_pep, the rate of peptide bond formation, for the many different aa-tRNAs studied (Fig. 5). Because the values differ over such a large range, both rates are plotted on a logarithmic scale. All five of the WT tRNAs aminoacylated with their cognate amino acids cluster in a narrow region of k_off, consistent with previous studies showing that E. coli aa-tRNAs bind EF-Tu with similar affinity(19). The slightly greater variance in k_pep for these tRNAs is primarily due to their lack of posttranscriptional modifications in the anticodon loop that influence their activity (27, 32). When aa-tRNAs were modified such that k_off was slower than WT, the early decoding steps were not affected, but their k_pep values were reduced. These slow k_pep values are best explained by the slow release of these hyperstabilized aa-tRNAs from EF-Tu•GDP becoming rate-limiting for peptide bond formation. Experiments using the E378A mutation of EF-Tu to revert the slow k_pep values in the expected sequence-selective manner strongly support this conclusion. In contrast, when the T stem of Val-tRNA^Val was mutated such that k_off was faster than the WT sequence (variants W1 to W3), all of the steps in decoding were unaffected, provided that sufficiently high EF-Tu concentrations were present to permit the ternary complex to form. Unlike for the tight tRNAs, k_pep values for the weak aa-tRNAs were not correlated with the value of k_off, indicating that EF-Tu release does not limit k_pep for any of them. Because the native aa-tRNAs all have k_pep values similar to the weak tRNAs, it appears that their sequences have evolved to release from EF-Tu at a rate that is either faster or similar to the rate-limiting step.

Fig. 5.

Comparison of k_off and k_pep for all aa-tRNAs tested. Rates of dissociation from E. coli EF-Tu for: WT aa-tRNAs ▪, Val-tRNA^Val mutants ♦, T1 mutants (tRNAs Ala, Gly, Phe, and Tyr) ▴, and misacylated tRNAs ● are compared to rates of peptide bond formation of the same aa-tRNAs. The solid line is a linear fit to all tight-binding mutants with k_off < 5 × 10^-4 s^-1, while the dotted line is a linear fit to WT and weaker-binding mutants.

An interesting feature of Fig. 5 is that k_pep and k_off are proportional for all the tight-binding aa-tRNA mutations tested. While the k_off data in Fig. 5 were obtained at 4 °C to permit the weaker aa-tRNAs to be measured, k_off values for a selected number of tight tRNAs were determined at the same conditions used for k_pep (Table S1). These experiments show that k_off and k_pep remain proportional for the different aa-tRNAs, but k_pep is consistently about 100-fold faster than k_off. While both rates reflect dissociation of the tight aa-tRNAs from the protein, the structures of the complexes are very different. In the ribosome-bound complex, EF-Tu is in the GDP form and the aa-tRNA is in a distorted, presumably high-energy conformation, which are both expected to destabilize the protein–tRNA interface (18, 38). However, the proportional effects on k_off and k_pep for both a substitution of the esterified amino acid and several T-stem mutations suggest that the entire interface between EF-Tu and aa-tRNA is thermodynamically similar both on and off the ribosome. Additional protein and aa-tRNA mutations will be needed to confirm this point.

It is striking that the proportionality between k_off and k_pep is maintained over a wide range of values with several tRNA species whose affinities were increased with different T-stem sequences and esterified amino acids. This indicates that, despite the differences in the two structures, the specificity of EF-Tu for the different esterified amino acids and tRNA bodies previously established for the free ternary complex also occurs at the release step on the ribosome. This explains why aa-tRNAs have evolved to have a uniform affinity for EF-Tu. Two different steps in the decoding pathway combine to constrain the affinity to a narrow range. Each aa-tRNA must initially bind EF-Tu tightly enough to efficiently form the ternary complex but also must bind weakly enough to be released from the protein after GTP hydrolysis during decoding. As a result, the WT aa-tRNAs have evolved to be in the general region of Fig. 5 where the affinity is as tight as possible without slowing k_pep. A mutation of any tRNA that either tightens or weakens its affinity to EF-Tu will impact translational performance and presumably be selected against. Since nearly all the EF-Tu residues that contact aa-tRNA are conserved among bacteria, it is likely that the conclusions made here for E. coli are generally applicable. Indeed, the thermodynamic effects of T-stem tRNA mutations determined here using E. coli EF-Tu are virtually identical to those previously determined for T. thermophilus (10, 15, 16). However, as discussed elsewhere, the sequences of individual tRNA species vary considerably among bacteria because multiple T-stem sequences can have a similar EF-Tu affinity (16, 39, 40).

By introducing the T-stem sequence of a tight tRNA into a weak tRNA, the resulting chimera may bind EF-Tu so tightly that k_pep becomes very slow. An extreme example of this was the T1 mutation of Tyr-tRNA^Tyr that gave a k_pep of 0.025 sec^-1. This chimera may be useful to more accurately place the EF-Tu release step in the decoding pathway. In addition, because a high-resolution structure is available that depicts the ternary complex just prior to release (18), detailed structure-function studies are now possible. By stabilizing binding to EF-Tu, the T1 chimeras may also be helpful for improving the efficiency of incorporating certain nonnatural amino acids into protein (41, 42). Finally, the decoding properties of the T1 chimera of Tyr-tRNA^Tyr are interesting to compare with the highly specialized system that has evolved to introduce selenocysteine into proteins. Like EF-Tu, its distant orthologue SelB has evolved to selectively bind its cognate Sec-tRNA^Sec in a manner that is specific for both the esterified amino acid and the tRNA body (43). Like the T1 tRNA^Tyr mutation, SelB binds Sec-tRNA^Sec extremely tightly. It will be interesting to see whether k_pep for selenocysteine incorporation is also unusually slow.

Materials and Methods

Materials.

Tight-coupled 70S ribosomes from E. coli MRE600 cells were prepared as described (44) and purified pellets were resuspended in buffer RB (50 mM HEPES [pH 7.0], 30 mM KCl, 70 mM NH₄Cl, 10 mM MgCl₂, and 1 mM DTT). Ribosomes were flash frozen and stored at -80 °C and activated as previously described (45). mRNA derivatives of the T4 gp32 mRNA fragment 5′-GGCAAGGAGGUAAAAAUGXXXGCACGU-3′, where XXX indicates the A site codon, were purchased from Dharmacon.

E. coli EF-Tu, EF-Tu(H84A) and EF-Tu(E378A) containing an N-terminal histidine tag and TEV protease sequence were over expressed in BL21-Gold (DE3) cells (Agilent) and purified by Ni-NTA chromatography as in (46). The concentration was determined using Pierce A660 protein assay using BSA as a standard.

Purified E. coli tRNA^fMet was purchased from Sigma Aldrich. Transcribed tRNAs were prepared as described (16) and stored in TE buffer (10 mM Tris-HCl [pH 7.6], 0.1 mM EDTA) at concentrations greater than 100 μM. Native E. coli tRNA^Val was purified by hybridization using biotinylated DNA oligonucleotides designed in (47). A 1 mL reaction of 4 mg/mL unfractionated E. coli tRNA (Roche) was heated to 65 °C in 10 mM EDTA for 10 min and 20X SSC buffer was added to a final concentration of 1X SSC (150 mM NaCl, 15 mM NaCitrate pH 7.0). The appropriate 3′ biotinylated DNA was added at a concentration of 17 μM and annealed at 65 °C for 15 min and at room temperature for 20 min. 500 μL streptavidin resin (Sigma Aldrich) in 1X SSC buffer was added and incubated at room temperature for 20 min to capture the probe. The resin was washed in a spin column three times with 1 mL 0.1X SSC, and the tRNA was eluted from the resin by the addition of 250 μL water at 65 °C for 10 min. The purified tRNA was then EtOH precipitated and additionally purified by 10% denaturing PAGE and stored in a TE buffer.

Aminoacylation of tRNAs with [³H]-amino acids (Amersham) was performed as in (16). 3′-[³²P] labeling of tRNA and aminoacylation was performed as previously described(33) with typical aminoacylation yields of > 70%. Misacylated and (G3-U70) derivatives were prepared as described (10, 11).

Methods.

The equilibrium binding affinity of ternary complexes to programmed E. coli ribosomes was determined essentially as described (27). Ternary complexes were formed with 150 nM EF-Tu(H84A) and < 0.7 nM [3′-³²P] aa-tRNA and added to a series of ribosome concentrations programmed with excess mRNA and tRNA^fmet to give final concentrations of 50 nM EF-Tu(H84A), < 0.23 nM aa-tRNA and from 600 nM to 0.5 uM encoded ribosomes. After incubation for 2 min at 20 °C, samples were filtered using a dual filter system and filters were quantified using a PhosphorImager (Storm 820). After subtraction of the counts on the lower filter due to ³²P deacylated tRNA, the data were fit to a simple binding isotherm using Kaleidagraph. Sample data is shown in Fig. 2A, and the data in Table 2 reports at least three separate measurements using different preparations of programmed ribosomes, fMet-tRNA^fMet and ternary complex.

The rate of [³H]-aa-tRNA dissociation from EF-Tu (k_off) was determined using an RNase protection assay as described (16) with the following exceptions: E. coli EF-Tu•GTP was activated for only 20 min (instead of 3 h for T. thermophilus EF-Tu), and reactions were carried out in RB buffer. The fraction of [³H]-aa-tRNA bound by EF-Tu over time was fit to a single exponential using Kaleidagraph. tRNA mutations with k_off values greater than 0.02 s^-1 or less than 0.0002 s^-1 were measured at multiple lower or higher NH₄Cl concentrations, and their k_off was extrapolated to 70 mM NH₄Cl present in RB (12). All measurements were performed in triplicate. k_off values were converted to ΔG° values using k_on = 1.1 × 10⁵ M^-1 s^-1 (19), (21).

Apparent rates of GTP hydrolysis were measured as in (27) (30). Ternary complex was formed in RB buffer by incubating 2 μM EF-Tu•GDP, 0.6 μM tRNA, 50 μM [γ-³²P]-GTP, 3 mM phophoenol pyruvate, 12 U/mL pyruvate kinase, 125 μM Valine, 4 mM ATP, 10 U/mL yeast pyrophosphatase, and 0.25 μM ValRS for 1 h at 37 °C and then purified through two P30 spin columns (Biorad). Ternary complex was mixed with equivalent volume of 1.0 to 8.0 μM programmed ribosomes in a rapid mixing device (Kintek), quenched in 40% formic acid at the desired time points, and separated using TLC (30). After subtraction of the excess EF-Tu•GTP present, the time course of the fraction of GTP hydrolyzed was fit to single exponential equation to obtain k_GTP. The concentration dependence was fit to the Michaelis–Menton equation to give k_GTPmax, the rate at saturation. Because each point on the k_GTPmax curve represents k_GTP hydrolysis determined using various batches of ribosomes, tRNA^fMet, and ternary complex, the errors are significant. However, errors are much less when a single set of components were used to obtain each k_GTP.

The rate of peptide bond formation was performed essentially as described (27). Programmed ribosomes were prepared by combining 2 μM ribosomes, 6 μM mRNA and 4 μM fMet-tRNA^fMet and mixed in a rapid mixing device (Kintek) with equivalent volume of 50 nM ³²P-labeled ternary complex (1 μM EF-Tu•GTP and 50 nM [3′ -³²P] aa-tRNA). Reactions were quenched at set time points with 5 mM NaOAc pH 5.2 100 mM EDTA, digested with P1 nuclease, and separated on PEI cellulose TLC plates as in (33). After correcting for the fraction of tRNA^fMet which was not charged or formylated, the fraction of dipeptide formed vs. time was fit to a single exponential by Kaleidagraph. All measurements were performed in triplicate.