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

Jared M. Schrader, Stephen J. Chapman, and Olke C. Uhlenbeck1

Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208

Contributed by Olke C. Uhlenbeck, February 8, 2011 (sent for review December 22, 2010)

To better understand why aminoacyl-tRNAs (aa-tRNAs) have release of the aa-tRNA from EF-Tu (18) reveals that although evolved to bind bacterial elongation factor Tu (EF-Tu) with uniform the forms multiple contacts with both the tRNA and affinities, mutant tRNAs with differing affinities for EF-Tu were EF-Tu, the overall arrangement of the three domains of EF-Tu assayed for decoding on Escherichia coli . At saturating and its interface with aa-tRNA is quite similar to that of the free EF-Tu concentrations, weaker-binding aa-tRNAs decode their cog- ternary complex. This suggests that the thermodynamic interplay nate codons similarly to wild-type tRNAs. However, tighter-binding between the esterified and the T-stem sequence may aa-tRNAs show reduced rates of bond formation due to also occur during decoding and provide an additional selective slow release from EF-Tu•GDP. Thus, the affinities of aa-tRNAs for pressure for uniform binding. This paper tests this possibility EF-Tu are constrained to be uniform by their need to bind tightly by examining the decoding properties of aa-tRNAs that have enough to form the ternary complex but weakly enough to release been altered to have either an increased or decreased affinity from EF-Tu during decoding. Consistent with available crystal for EF-Tu by changing the T-stem sequence or introducing a structures, the identity of the esterified amino acid and three base different esterified amino acid. 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. Results Val Derivatives of Escherichia coli tRNA GAC with Differing Affinities to E. coli Val he ternary complex of bacterial elongation factor Tu (EF-Tu), E. coli EF-Tu. The tRNA GAC species that decodes GUC/U TGTP and aminoacyl-tRNA (aa-tRNA) binds to the ribosome codons was chosen because its tRNA body has an intermediate and participates in a multistep decoding pathway in which GTP is affinity for EF-Tu (10), making it possible to both increase and hydrolyzed, EF-Tu•GDP is released, and the aa-tRNA enters the decrease its binding affinity by mutating the T stem. In addition, ribosomal A site (1–6). Although all elongator aa-tRNAs bind this tRNA is one of the few in E. coli that lacks posttranscrip- EF-Tu•GTP with similar affinities (7–9), studies with misacylated tional modifications in its anticodon hairpin, thereby maximizing tRNAs reveal that the shows substantial specificity for the likelihood that it will decode effectively as an unmodified both the esterified amino acid and the tRNA body (10–12). tRNA. Based upon the binding affinities of individual base-pair Phe The nearly uniform EF-Tu binding affinity observed for tRNAs substitutions in S. cerevisiae tRNA to T. thermophilus EF-Tu Val

A BIOCHEMISTRY acylated with their correct (cognate) amino acid occurs because (16), T-stem of tRNA GAC (Fig. 1 ) were designed the sequence of each tRNA has evolved to compensate for the to bind tighter (T1 to T3), weaker (W1 to W3), or similar (Ψ)to the wild-type (WT) tRNA sequence. After aminoacylation with variable thermodynamic contribution of the esterified amino 3 acid. Thus, weak-binding esterified amino acids such as glycine [ H]-valine, a ribonuclease protection assay was used to deter- and have corresponding tRNAs that bind the protein mine the dissociation rates of WT and the seven mutants of Val tightly, while tight-binding amino acids such as tyrosine or gluta- tRNA from E. coli EF-Tu•GTP at 4 °C (Fig. 1B, Table 1) mine have corresponding tRNAs that bind poorly. The crystal (19, 20). Dissociation rates for several tRNAs were also deter- k structure of Thermus aquaticus EF-Tu•GTP bound to Saccharo- mined at 20 °C, and off values were ninefold to 15-fold faster myces cerevisiae Phe-tRNAPhe (13) reveals that the protein than at 4 °C (Table S1). As previously observed with other Val primarily forms extensive interactions with the helical phospho- tRNAs,(12, 21, 22) the unmodified WT tRNA GAC bound Phe Val diester backbone of the acceptor and Tstems of tRNA . Recent EF-Tu very similarly to native, fully modified tRNA GAC consis- protein (14) and tRNA (15, 16) mutagenesis experiments indicate tent with the absence of modifications in the EF-Tu binding site. that much of the specificity is the result of interactions made The seven mutations showed the desired broad range of binding between three amino acids of EF-Tu and three adjacent base pairs affinity to E. coli EF-Tu that is similar to the range in affinities in the T stem (16). Additional mutagenesis experiments indicate observed for different tRNA bodies with T. thermophilus EF-Tu that the thermodynamic contribution of each of the three base (10, 16). T1 to T3 bound from 0.5 to 1.0 kcal∕mol tighter, W1 to pairs is independent of the others, making it possible to adjust W3 bound from 1.0 to 2.0 kcal∕mol weaker, and Ψ bound nearly Val the affinity of aa-tRNAs to EF-Tu in a predictable manner. the same as the WT tRNA . These measured values correlate Although a detailed structural and thermodynamic under- closely with the values predicted by adding the ΔΔG° values standing of how EF-Tu achieves uniform binding with different obtained from data measuring the binding of single base-pair aa-tRNAs is beginning to emerge, the underlying selective pres- substitutions in S. cerevisiae tRNAPhe to T. thermophilus EF-Tu sures that lead to uniform binding are less clear. While aa-tRNAs (Fig. 1C). This not only supports the notion that the thermody- must bind EF-Tu tightly enough to participate in translation, the namic contributions of the three T-stem base pairs are indepen- high intracellular concentration of EF-Tu (17) ensures that they dent but indicates that both E. coli and T. thermophilus EF-Tu do not significantly compete with one another for the protein. interact with aa-tRNAs in a thermodynamically similar manner. It therefore seems unlikely that a minimum threshold binding

affinity would provide sufficient selective pressure to ensure the Author contributions: J.M.S. and O.C.U. designed research; J.M.S. and S.J.C. performed observed uniform binding properties. Additional selective pres- research; S.J.C. contributed new reagents/analytic tools; J.M.S. analyzed data; and sure could potentially come at the step in translation where J.M.S. and O.C.U. wrote the paper. aa-tRNAs are released from EF-Tu•GDP prior to peptide bond The authors declare no conflict of interest (such as defined by PNAS policy). formation. This could limit the affinity to a maximal threshold, 1To whom correspondence should be addressed. E-mail: [email protected]. thereby selecting for uniform binding. The recent crystal struc- This article contains supporting information online at www.pnas.org/lookup/suppl/ ture of a ribosome-bound ternary complex trapped just before doi:10.1073/pnas.1102128108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1102128108 PNAS ∣ March 29, 2011 ∣ vol. 108 ∣ no. 13 ∣ 5215–5220 Downloaded by guest on October 1, 2021 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-tRNAVal on E. coli ribosomes.

somes differed by about twofold between the four Val-tRNAVal derivatives tested. When the rates of GTP hydrolysis were plotted as a function of ribosome concentration (Fig. 3C), values of K1∕2 k and GTPmax (Table 2) also differ by about twofold. Thus, either increasing (T1) or decreasing (W1) the affinity of Val-tRNAVal 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. k Val The tRNA modifications also had little effect on GTP. Fig. 1. Engineering Val-tRNA mutants with altered EF-Tu binding k Val The final assay measures pep, the rate of ribosome-catalyzed affinities. (A) E. coli tRNA GAC with mutated T-stem base pairs in box. T-stem sequences of WT and seven mutant tRNA transcripts are on the right. (B)In- formation of fMetVal-tRNAVal. This final step in decoding k ● 12 × 10−4 −1 ♦ dividual rates of dissociation ( off) for WT ( s ), W1 encompasses the release of both inorganic phosphate and −4 −1 −4 −1 −4 −1 (170 × 10 s ), T1 ▪ (2.0 × 10 s ), and native ▴ (5.5 × 10 s ) Val • Val Val-tRNA from EF-Tu GDP, the accommodation of the Val-tRNA from E. coli EF-Tu at 0 °C in RB buffer using the ribonuclease Val protection assay. Means and standard errors from multiple determinations Val-tRNA into the A site and the formation of the peptide are in Table 1. (C) Comparison of the free energies of binding (ΔG°) between bond. It remains uncertain which one of these substeps limits Val k Val-tRNA T-stem mutants to E. Coli EF-Tu with measured ▪ or calculated the value of pep for wild-type aa-tRNAs, and it may depend upon ♦ free energies of the same mutants in S. cerevisiae Phe-tRNAPhe binding the assay conditions and the aa-tRNA that are used (28, 31, 32). to T. thermophilus EF-Tu. The line corresponds to a least square fit with a k ′ 32 Here pep is measured with ternary complexes containing 3 -[ P]- slope of 1.1. labeled Val-tRNAVal using a P1 nuclease assay (33). The ternary Decoding Properties of tRNAVal T-stem Mutants. Three quantitative complexes containing native tRNAVal, the WT, and W1 deriva- k D assays were used to evaluate the ability of ternary complexes tives all show similar pep values and extents of reaction (Fig. 3 , made with native tRNAVal and the unmodified WT, T1, and Table 2). Strikingly, the ternary complex containing tight-binding E. coli Val k W1 tRNAs to decode the GUC codon on ribosomes con- T1 tRNA is fully active but shows a 14-fold slower pep value fMet k taining fMet-tRNA in the P site (Fig. 2, Table 2). The experi- than WT. This slow pep can potentially be explained by its slow ments were performed at 20 °C in a buffer containing 10 mM • k release from EF-Tu GDP (Fig. 2). To further test this, pep values MgCl2 to ensure that the unmodified tRNAs were fully folded Val – for the remaining five tRNA derivatives were determined (23 25). The first assay measures the equilibrium binding con- (Table 1). The values of k for both the weaker-binding W2 stant of the ternary complex to ribosomes by using the active site pep and the very weak-binding W3 mutation are similar mutant of EF-Tu(H84A) to prevent GTP hydrolysis (26, 27). The to WT, although higher concentrations of EF-Tu are required resulting equilibrium constant reflects the initial binding, codon in the latter case to achieve high levels of dipeptide. The Ψ muta- recognition, and GTPase steps of the decoding path- tion, which binds similarly to WT, also shows a similar k value. way (28). As shown in Fig. 3A, all of the tRNAVal derivatives show pep In contrast, T2 and T3, which both bind to EF-Tu more tightly, very similar Kd values. Thus, neither the tRNA modifications nor are fully active but show significantly slower k values. Taken the T-stem mutations significantly alter the initial interaction of pep the ternary complex with the ribosome. together, the data indicate that while removing the modifications The second assay uses ternary complexes made with γ-[32P]- or weakening the affinity to EF-Tu has little effect on the rate of GTP to measure the rate of GTP hydrolysis as a function of peptide bond formation, tightening the interaction with EF-Tu encoded ribosome concentration. This rate reflects a conforma- clearly reduces it. – tional change in EF-Tu that occurs before bond cleavage (28 30). k B μ Rescuing the Slow pep with a Sequence-Specific EF-Tu Mutation. If As shown in Fig. 3 , the rate of GTP hydrolysis at 2.5 M ribo- k the reduced pep values observed with the three hyperstabilized Val k k Val tRNA derivatives are the result of slow release from EF-Tu• Table 1. off and pep values of Val-tRNA mutants GDP, it should be possible to reverse this effect with an EF-Tu Free energy of mutation that destabilizes tRNA binding. To test this, we made Rate of aa-tRNA aa-tRNA Binding Rate of peptide use of a contact between EF-Tu and tRNA that stabilizes the dissociation from to EF-Tu ΔG° bond formation 104 × k ð −1Þ ∕ k ð −1Þ Val tRNA EF-Tu off s (kcal mol) pep s Table 2. Performance of Val-tRNA mutants in decoding − Native 5.6 (±) 0.4 10.4 (±) 0.1 3.3 (±) 0.4 A/T site Maximal rate Half maximal [70S] − WT 13 (±) 4.0 9.9 (±) 0.3 4.5 (±) 0.7 affinity of GTP hydrolysis for GTP hydrolysis T1 2.0 (±) 0.5 −10.9 (±) 0.3 0.32 (±) 0.02 −1 tRNA Kd (nM) k (s ) K1∕2 (μM) T2 3.4 (±) 1.3 −10.6 (±) 0.2 0.55 (±) 0.04 GTPmax T3 5.3 (±) 0.5 −10.4 (±) 0.1 1.4 (±) 0.2 Native 1.1 (±) 0.3 27 (±) 9.7 2.6 (±) 1.9 Ψ 22 (±) 5.0 −9.6 (±) 0.2 4.5 (±) 0.8 WT 0.8 (±) 0.2 27 (±) 7.4 2.7 (±) 1.4 W1 110 (±) 6.7 −8.8 (±) 0.1 5.4 (±) 2.3 T1 0.5 (±) 0.2 41 (±) 13 2.2 (±) 1.4 W2 130 (±) 39 −8.6 (±) 0.3 6.0 (±) 2.5 W1 2.1 (±) 0.7 28 (±) 8.3 1.6 (±) 1.2 − W3* 950 (±) 210 7.6 (±) 0.2 3.3 (±) 0.1 K k Standard errors for A/T site d and pep are calculated from at least three Standard errors are calculated from at least three independent independent determinations. k K – determinations. Standard errors for GTPmax and 1∕2 are fit to the Michaelis Menton k k * off value extrapolated from lower NH4Cl concentrations. equation with at least six GTPapparent determinations per curve.

5216 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1102128108 Schrader et al. Downloaded by guest on October 1, 2021 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). To confirm that the E378A mutation of E. coli EF-Tu shows the expected specificity for binding to the tRNAVal derivatives, k off values were determined for ternary complexes of this mutant protein with WT, Ψ, and the three tight tRNAVal derivatives k Val (Table 3). The off for the WT Val-tRNA was not significantly affected by the E378A mutation, consistent with the fact that it contains a U51-A63 . However, the Ψ, T1, and T2 derivatives, which contain G51-C63, dissociate fivefold to six- fold 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. k Ψ Val pep values for WT, , T1, T2, and T3 versions of Val-tRNA were then determined using ternary complexes containing EF-Tu k (E378A) (Table 3). The value of pep for the WT tRNA with the −1 Fig. 3. Activities of Val-tRNAVal mutants in decoding. Individual determina- mutant EF-Tu (4.4 s ) was identical to that observed using WT −1 tions of the performance of WT ●,W1♦,T1▪, and native ▴ Val-tRNAVal on EF-Tu (4.5 s ). Because WT Val-tRNAVal has a U51-A63 encoded ribosomes at 20 °C in RB using three assays. Means and standard base pair and is not stabilized by the E378 residue, this establishes errors of multiple determinations are in Table 2. (A) Binding affinities of that EF-Tu(E378A) is fully active in decoding. However, the H84A (GTPase deficient) EF-Tu ternary complexes to the ribosomal A/T site k 0 23 pep values for the tightly binding T1 and T2 tRNAs with EF- using < . nM ternary complex. Lines are best fit simple binding curves with −1 −1 K ¼ 0 8 γ 32 Tu(E378A) are 2.7 s and 3.3 s , respectively. Because these d . nM, 1.5 nM, 0.7 nM, and 1.4 nM, respectively. (B) Rates of -[ P]- −1 −1 k μ 0 32 0 55 GTP hydrolysis ( GTP) at 200 nM ternary complex and 2.5 M ribosomes. Lines values are substantially faster than the . s and . s k ¼ 10 −1 16 −1 7 5 −1 11 −1 are best fit single exponential rates with GTP s , s , . s , s , observed with WT EF-Tu, it is clear that the EF-Tu mutation that respectively. (C) Ribosome saturation curve fit to the Michaelis–Menten equa- weakens binding to the hyperstabilized T1 and T2 tRNAVal K k tion for 1∕2 and GTPmax(see Table 2). (D) Rates of peptide bond formation derivatives also reverts their abnormally slow k values. This k μ pep ( pep) measured at 25 nM ternary complex and 1 M ribosomes. Lines k ¼ 5 7 −1 8 1 −1 0 34 −1 3 8 −1 experiment shows that the hydrogen bond between E378 and are single exponential fits with pep . s , . s , . s , and . s , respectively. G63 in the ribosome-bound ternary complex contributes to its stability and thereby influences the rate of release of an aa-tRNA BIOCHEMISTRY interaction in a sequence-specific manner (15). In the ternary into the A site. Phe k complex structure of E. coli EF-Tu with E. coli Phe-tRNA Two critical control experiments are the pep measurements of (PDB ID code 1OB2), E378 makes a hydrogen bond with the the Ψ and T3 tRNAs with EF-Tu(E378A). Despite the fact that Ψ amino group of G63 (Fig. 4A). Mutation of the homologous contains a G51-C63 and thus binds more weakly to the E378A k Ψ 3 7 −1 E390 in T. thermophilus EF-Tu to an alanine reduced the binding protein, pep for with EF-Tu(E378A) ( . s ) is similar to WT −1 affinity of the protein to tRNAs containing a G at either position EF-Tu (4.5 s ). This experiment shows that the E378A mutation k 51 or 63 but not to tRNAs lacking a G (15). A similar hydrogen only stimulates pep when the tRNA is hyperstabilized for EF-Tu k bond is observed between E390 of T. thermophilus EF-Tu and G63 binding. The second control shows that the slow pep observed −1 of E. coli Thr-tRNAThr in the structure of the ternary complex for the T3 tRNAVal with WT EF-Tu (1.4 s ) remains slow with −1 bound to T. thermophilus ribosomes in the presence of kirromycin EF-Tu(E378A) (0.6 s ). Because the A51-C63 pair present in k (Fig. 4B) (18). Because this structure depicts EF-Tu after GTP T3 is not stabilized by E378, its pep is unaffected by the E378A mutation. This control shows that when the ternary complex is k hyperstabilized in a different way, the slow pep no longer requires the stabilizing contact using E378. Taken together, the experi- k ments with EF-Tu(E378A) confirm that the slow pep values observed for the hyperstabilized tRNAVal derivatives are caused by the slow release from EF-Tu.

k k Val Table 3. off and pep values of Val-tRNA mutants with EF-Tu (E378A) Free energy of Rate of aa-tRNA aa-tRNA binding Rate of peptide dissociation from to EF-Tu bond formation 104 × k −1 Δ ∕ k −1 tRNA EF-Tu off (s ) G° (kcal mol) pep (s ) WT 18 (±) 8.9 −9.7 (±) 0.5 4.4 (±) 1.5 Fig. 4. The EF-Tu•tRNA interface maintains similar interactions on the ribo- T1 10 (±) 3.7 −10.0 (±) 0.4 2.7 (±) 0.4 some. (A) Cocrystal structure of E. coli EF-Tu bound to S. cerevisiae T2 19 (±) 6.7 −9.7 (±) 0.4 3.1 (±) 0.3 Phe-tRNAPhe with kirromycin (PDB) highlighting the interactions made be- T3 5.2 (±) 0.4 −10.4 (±) 0.1 0.59 (±) 0.3 tween E378, Q329, and T338 10B2 (E390, Q341, T350 in T. thermophilus) with Ψ 140 (±) 36 −8.6 (±) 0.3 3.7 (±) 0.6 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-tRNAThr stalled on the ribosome with Standard errors are calculated from at least three independent kirromycin post GTP hydrolysis (PDB ID code 2WRN) (18). determinations.

Schrader et al. PNAS ∣ March 29, 2011 ∣ vol. 108 ∣ no. 13 ∣ 5217 Downloaded by guest on October 1, 2021 k k Contribution of the Esterified Amino Acid to pep. While the above 100-fold difference in their off values, supporting the view that experiments establish that the sequence of the T stem can influ- the compensatory relationship between the esterified amino acid ence the rate of release from EF-Tu•GDP during decoding, it is and tRNA body observed for the free ternary complex occurs in not clear whether the identity of the esterified amino acid can an identical way during decoding. influence this step in a manner similar to what is observed in Although the above data strongly suggests that the identity of the formation of the ternary complex. In the ribosome-bound the esterified amino acid can contribute to the rate of release ternary complex (18), the position of the esterified amino acid from EF-Tu•GDP during decoding, experiments with misacy- is different than in the free ternary complex, so it is uncertain lated-tRNAs are needed to show this directly. We made use of whether the thermodynamic effect of the amino acid would be the observation that tRNAVal can be misacylated by high concen- k Val similar. One way to detect an effect of the amino acid on pep trations of -PheRS, and the resulting Phe-tRNA binds is to introduce the hyperstabilizing T1 sequence into other E. coli EF-Tu twofold to threefold tighter than Val-tRNAVal (10). Table5 k tRNAs. Because the specificity of EF-Tu for tRNA is primarily shows that the pep values of WT and T1 are 4.3- and 2.2-fold defined by the T-stem sequence (16), the T1 chimeras are slower when phenylalanylated than when valylated, which agrees expected to have a similar tight affinity to EF-Tu•GTP, and any reasonably well with the twofold to threefold difference reported k k difference in k should primarily reflect differences in the in off (10). In contrast, the pep of the phenylalanylated W1 tRNA off −1 −1 esterified amino acid. This T1 “upgrade” mutation was intro- (5.1 s ) is the same as when it is valylated (5.4 s ) because the E. coli Gly Ala relatively large (1.1 kcal∕mol) destabilizing effect of the W1 mu- duced into the bodies of tRNA CCC, tRNA GGC, Phe Tyr tation more than offsets the relatively small (0.5 kcal∕mol) effect tRNA GAA, and tRNA GUA, and the resulting chimeras were k acylated with their corresponding cognate amino acids. off values of introducing the phenylalanine, so the aa-tRNA is not hyperst- for the chimeras were compared with the WT aa-tRNAs in abilized. In order to show that misacylation can also lead to a k Tyr Table 4. In general, the T1 mutation stabilized the binding of each faster pep, the acceptor stem of the T1 derivative of tRNA aa-tRNA to EF-Tu•GTP in the expected manner. Because was mutated to contain a G3-U70 base pair so it could be alany- k tRNAGly and tRNAAla are tight-binding tRNA sequences lated by AlaRS(37). As shown in Table 5, pep for this Ala- CCC GGC Tyr (10), their T1 chimeras only bound EF-Tu twofold and fourfold tRNA is 61-fold faster than when esterified with the tight- tighter than their WT counterparts. The T1 chimera of binding tyrosine. These experiments with misacylated tRNAs Phe confirm that the esterified amino acid contributes to the rate tRNA GAA, an intermediate-binding tRNA, binds 56-fold tigh- ter than the WT tRNAPhe. For the T1 chimera of the very weak- of release from EF-Tu•GDP during decoding in a compensatory Tyr manner that is very similar to that observed for the formation of binding tRNA GAC, dissociation from EF-Tu GTP was too slow k ternary complex. to obtain a reliable off , even when the ionic strength was in- creased to 3 M NH4Cl or the temperature raised to 20 °C. k Tyr Discussion An estimated off for the T1 Tyr-tRNA , calculated based on Tyr Several conclusions of this paper can be conveniently summar- its sequence (16), was 62-fold tighter than WT tRNA . k k ized by plotting off , the rate of dissociation of aa-tRNA from pep values for the eight tRNAs are also shown in Table 4. The • k k EF-Tu GTP, versus pep; the rate of peptide bond formation, four unmodified WTaa-tRNAs had pep values that were reason- −1 for the many different aa-tRNAs studied (Fig. 5). Because the ably similar to WT tRNAVal (4.5 s ). The slightly slower rates for values differ over such a large range, both rates are plotted on tRNATyr (1.4s−1) and tRNAPhe (2.3 s−1) probably reflect GUA GAA a logarithmic scale. All five of the WT tRNAs aminoacylated the absence of modifications in the anticodon hairpin, which is k with their cognate amino acids cluster in a narrow region of off, known to weaken ribosome binding (35). The slightly slower rate E. coli Gly 1 4 −1 consistent with previous studies showing that aa-tRNAs for tRNA CCC ( . s ) probably reflects the lower pKa of gly- k bind EF-Tu with similar affinity(19). The slightly greater variance cine (36). The pep values for the four T1 derivatives were all in k for these tRNAs is primarily due to their lack of posttran- slower than the corresponding WT tRNAs and correlated closely pep k k scriptional modifications in the anticodon loop that influence with their off rates. Thus, pep values for the T1 derivatives of Gly Ala their activity (27, 32). When aa-tRNAs were modified such that Gly-tRNA CCC and Ala-tRNA GCC were only twofold to k k off was slower than WT, the early decoding steps were not threefold slower than the WT tRNAs. However, pep of the T1 k k Phe affected, but their pep values were reduced. These slow pep mutant of Phe-tRNA GAA was 13-fold slower than that of values are best explained by the slow release of these hyperstabil- WT Phe-tRNAPhe. Finally, the T1 mutation of Tyr-tRNATyr • k 0 025 −1 ized aa-tRNAs from EF-Tu GDP becoming rate-limiting for shows a very slow pep of . s , which is 57-fold slower than peptide bond formation. Experiments using the E378A mutation Tyr k measured for WT Tyr-tRNA . The 104-fold range in pep for the k of EF-Tu to revert the slow pep values in the expected sequence- T1 mutations correlates remarkably well with the greater than selective manner strongly support this conclusion. In contrast, when the T stem of Val-tRNAVal was mutated such that k Table 4. k and k values of WT and T1 mutations off off pep was faster than the WT sequence (variants W1 to W3), all of Rate of the steps in decoding were unaffected, provided that sufficiently aa-tRNA Free energy of Rate of high EF-Tu concentrations were present to permit the ternary k dissociation aa-tRNA binding peptide bond complex to form. Unlike for the tight tRNAs, pep values for k from EF-Tu to EF-Tu formation the weak aa-tRNAs were not correlated with the value of off , 104 × k -1 Δ ∕ k −1 k tRNA off (s ) G° (kcal mol) pep (s ) indicating that EF-Tu release does not limit pep for any of them. k Ala-tRNAAla WT 21 (±) 2.3 −9.7 (±) 0.1 6.8 (±) 0.6 Because the native aa-tRNAs all have pep values similar to Ala-tRNAAla T1 4.8 (±) 0.7 −10.5 (±) 0.2 2.6 (±) 0.1 the weak tRNAs, it appears that their sequences have evolved to Gly-tRNAGly WT 29 (±) 1.7 −9.5 (±) 0.1 1.4 (±) 0.6 Gly k Gly-tRNA T1 17 (±) 1.3 −9.8 (±) 0.1 0.81 (±) 0.3 Table 5. pep of misacylated tRNAs Phe Phe-tRNA WT 35 (±) 7.6 −9.4 (±) 0.1 2.3 (±) 1.1 −1 tRNA Rate of peptide bond formation k (s ) Phe-tRNAPhe T1 0.6* (±) 0.1 −11.6 (±) 0.2 0.18 (±) 0.11 pep Tyr-tRNATyr WT 9.9 (±) 0.1 −10.1 (±) 0.1 1.4 (±) 0.5 Phe-tRNAVal WT 1.5 (±) 0.7 Tyr-tRNATyr T1 0.16† −12.3 0.025 (±) 0.012 Phe-tRNAVal T1 0.16 (±) 0.04 Phe-tRNAVal W1 5.1 (±) 0.8 *Beyond limit of assay, estimated value. Tyr †k Ala-tRNA T1 1.4 (±) 0.4 off value extrapolated from higher NH4Cl concentrations. Standard errors are calculated from at least three independent Standard errors are calculated from at least three independent determinations. determinations.

5218 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1102128108 Schrader et al. Downloaded by guest on October 1, 2021 T. thermophilus (10, 15, 16). However, as discussed elsewhere, the sequences of individual tRNA species vary considerably among 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 Tyr k 0 025 −1 mutation of Tyr-tRNA that gave a pep of . sec . This chimera may be useful to more accurately place the EF-Tu release step in the decoding pathway. In addition, because a high-resolu- tion 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-tRNATyr are inter- esting to compare with the highly specialized system that has k k evolved to introduce into . Like EF-Tu, Fig. 5. Comparison of off and pep for all aa-tRNAs tested. Rates of ▪ Val ♦ its distant orthologue SelB has evolved to selectively bind its dissociation from E. coli EF-Tu for: WT aa-tRNAs , Val-tRNA mutants , Sec T1 mutants (tRNAs Ala, Gly, Phe, and Tyr) ▴, and misacylated tRNAs ● cognate Sec-tRNA in a manner that is specific for both the are compared to rates of peptide bond formation of the same aa-tRNAs. esterified amino acid and the tRNA body (43). Like the T1 k 5× Tyr Sec The solid line is a linear fit to all tight-binding mutants with off < tRNA mutation, SelB binds Sec-tRNA extremely tightly. −4 −1 k 10 s , while the dotted line is a linear fit to WT and weaker-binding It will be interesting to see whether pep for selenocysteine incor- mutants. poration is also unusually slow. release from EF-Tu at a rate that is either faster or similar to the Materials and Methods rate-limiting step. Materials. Tight-coupled 70S ribosomes from E. coli MRE600 cells were k k An interesting feature of Fig. 5 is that pep and off are propor- prepared as described (44) and purified pellets were resuspended in buffer tional for all the tight-binding aa-tRNA mutations tested. While RB (50 mM HEPES [pH 7.0], 30 mM KCl, 70 mM NH4Cl, 10 mM MgCl2,and k 1 mM DTT). Ribosomes were flash frozen and stored at −80 °C and activated the off data in Fig. 5 were obtained at 4 °C to permit the weaker k as previously described (45). mRNA derivatives of the T4 gp32 mRNA frag- aa-tRNAs to be measured, off values for a selected number of ′ ′ tight tRNAs were determined at the same conditions used for ment 5 -GGCAAGGAGGUAAAAAUGXXXGCACGU-3 , where XXX indicates k k k the A site codon, were purchased from Dharmacon. pep (Table S1). These experiments show that off and pep remain E. coli EF-Tu, EF-Tu(H84A) and EF-Tu(E378A) containing an N-terminal k proportional for the different aa-tRNAs, but pep is consistently histidine tag and TEV protease sequence were over expressed in BL21-Gold k about 100-fold faster than off . While both rates reflect dissocia- (DE3) cells (Agilent) and purified by Ni-NTA chromatography as in (46). The

tion of the tight aa-tRNAs from the protein, the structures of the concentration was determined using Pierce A660 protein assay using BSA as a BIOCHEMISTRY complexes are very different. In the ribosome-bound complex, standard. EF-Tu is in the GDP form and the aa-tRNA is in a distorted, Purified E. coli tRNAfMet was purchased from Sigma Aldrich. Transcribed presumably high-energy conformation, which are both expected 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. to destabilize the protein tRNA interface (18, 38). However, the Val k k Native E. coli tRNA was purified by hybridization using biotinylated proportional effects on off and pep for both a substitution of the DNA designed in (47). A 1 mL reaction of 4 mg∕mL unfrac- esterified amino acid and several T-stem mutations suggest that tionated E. coli tRNA (Roche) was heated to 65 °C in 10 mM EDTA for 10 min the entire interface between EF-Tu and aa-tRNA is thermodyna- and 20X SSC buffer was added to a final concentration of 1X SSC (150 mM mically similar both on and off the ribosome. Additional protein NaCl, 15 mM NaCitrate pH 7.0). The appropriate 3′ biotinylated DNA was and aa-tRNA mutations will be needed to confirm this point. added at a concentration of 17 μM and annealed at 65 °C for 15 min and k k at room temperature for 20 min. 500 μL streptavidin resin (Sigma Aldrich) It is striking that the proportionality between off and pep is maintained over a wide range of values with several tRNA species 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 whose affinities were increased with different T-stem sequences times with 1 mL 0.1X SSC, and the tRNA was eluted from the resin by the and esterified amino acids. This indicates that, despite the differ- addition of 250 μL water at 65 °C for 10 min. The purified tRNA was then ences in the two structures, the specificity of EF-Tu for the dif- EtOH precipitated and additionally purified by 10% denaturing PAGE and ferent esterified amino acids and tRNA bodies previously stored in a TE buffer. established for the free ternary complex also occurs at the release Aminoacylation of tRNAs with [3H]-amino acids (Amersham) was step on the ribosome. This explains why aa-tRNAs have evolved performed as in (16). 3′-[32P] labeling of tRNA and aminoacylation was to have a uniform affinity for EF-Tu. Two different steps in the performed as previously described(33) with typical aminoacylation yields of >70%. Misacylated tRNAVal and tRNATyr (G3-U70) derivatives were decoding pathway combine to constrain the affinity to a narrow GAC GUA prepared as described (10, 11). range. Each aa-tRNA must initially bind EF-Tu tightly enough to efficiently form the ternary complex but also must bind weakly Methods. The equilibrium binding affinity of ternary complexes to pro- enough to be released from the protein after GTP hydrolysis grammed E. coli ribosomes was determined essentially as described (27). during decoding. As a result, the WT aa-tRNAs have evolved Ternary complexes were formed with 150 nM EF-Tu(H84A) and <0.7 nM to be in the general region of Fig. 5 where the affinity is as tight [3′-32P] aa-tRNA and added to a series of ribosome concentrations pro- k fmet as possible without slowing pep. A mutation of any tRNA that grammed with excess mRNA and tRNA to give final concentrations of either tightens or weakens its affinity to EF-Tu will impact trans- 50 nM EF-Tu(H84A), <0.23 nM aa-tRNA and from 600 nM to 0.5 uM encoded lational performance and presumably be selected against. Since ribosomes. After incubation for 2 min at 20 °C, samples were filtered using a nearly all the EF-Tu residues that contact aa-tRNA are conserved 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 among bacteria, it is likely that the conclusions made here for E. coli tRNA, the data were fit to a simple binding isotherm using Kaleidagraph. are generally applicable. Indeed, the thermodynamic Sample data is shown in Fig. 2A, and the data in Table 2 reports at least effects of T-stem tRNA mutations determined here using E. coli three separate measurements using different preparations of programmed EF-Tu are virtually identical to those previously determined for ribosomes, fMet-tRNAfMet and ternary complex.

Schrader et al. PNAS ∣ March 29, 2011 ∣ vol. 108 ∣ no. 13 ∣ 5219 Downloaded by guest on October 1, 2021 3 k • The rate of [ H]-aa-tRNA dissociation from EF-Tu ( off) was determined TLC (30). After subtraction of the excess EF-Tu GTP present, the time course using an RNase protection assay as described (16) with the following excep- of the fraction of GTP hydrolyzed was fit to single exponential equation to • k – tions: E. coli EF-Tu GTP was activated for only 20 min (instead of 3 h for T. obtain GTP. The concentration dependence was fit to the Michaelis Menton k thermophilus EF-Tu), and reactions were carried out in RB buffer. The fraction equation to give GTPmax, the rate at saturation. Because each point on the 3 k k of [ H]-aa-tRNA bound by EF-Tu over time was fit to a single exponential GTPmax curve represents GTP hydrolysis determined using various batches k 0 02 −1 fMet using Kaleidagraph. tRNA mutations with off values greater than . s of ribosomes, tRNA , and ternary complex, the errors are significant. How- −1 or less than 0.0002 s were measured at multiple lower or higher NH4Cl ever, errors are much less when a single set of components were used to k k concentrations, and their off was extrapolated to 70 mM NH4Cl present obtain each GTP. k in RB (12). All measurements were performed in triplicate. off values were The rate of peptide bond formation was performed essentially as de- Δ k ¼ 1 1 × 105 −1 −1 μ converted to G° values using on . M s (19), (21). scribed (27). Programmed ribosomes were prepared by combining 2 M Apparent rates of GTP hydrolysis were measured as in (27) (30). Ternary ribosomes, 6 μM mRNA and 4 μM fMet-tRNAfMet and mixed in a rapid mixing complex was formed in RB buffer by incubating 2 μM EF-Tu•GDP, 0.6 μM device (Kintek) with equivalent volume of 50 nM 32P-labeled ternary complex tRNA, 50 μM[γ-32P]-GTP, 3 mM phophoenol pyruvate, 12 U∕mL pyruvate (1 μM EF-Tu•GTP and 50 nM [3′ -32P] aa-tRNA). Reactions were quenched at , 125 μM Valine, 4 mM ATP, 10 U∕mL yeast pyrophosphatase, and set time points with 5 mM NaOAc pH 5.2 100 mM EDTA, digested with P1 0.25 μM ValRS for 1 h at 37 °C and then purified through two P30 spin nuclease, and separated on PEI cellulose TLC plates as in (33). After correcting columns (Biorad). Ternary complex was mixed with equivalent volume of for the fraction of tRNAfMet which was not charged or formylated, the frac- 1.0 to 8.0 μM programmed ribosomes in a rapid mixing device (Kintek), tion of dipeptide formed vs. time was fit to a single exponential by Kaleida- quenched in 40% formic acid at the desired time points, and separated using graph. All measurements were performed in triplicate.

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5220 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1102128108 Schrader et al. Downloaded by guest on October 1, 2021