Important for tRNA Selection by the Ribosome
Prashant K. Khade, Xinying Shi and Simpson Joseph
Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093–0314, USA
Correspondence to Simpson Joseph: 4102 Urey Hall, Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093–0314, [email protected]
http://dx.doi.org/10.1016/j.jmb.2013.02.038 Edited by D. E. Draper
Abstract
Accurate tRNA selection by the ribosome is essential for the synthesis of functional proteins. Previous structural studies indicated that the ribosome distinguishes between cognate and near-cognate tRNAs by monitor-ing the geometry of the codon–anticodon helix in the decoding center using the universally conserved 16S ribosomal RNA bases G530, A1492 and A1493. These bases form hydrogen bonds with the 2′-hydroxyl groups of the codon–anticodon helix, which are expected to be disrupted with a near-cognate codon–anticodon helix. However, a recent structural study showed that G530, A1492 and A1493 form hydrogen bonds in a manner identical with that of both cognate and near-cognate codon–anticodon helices. To understand how the ribosome discriminates between cognate and near-cog-nate tRNAs, we made 2′-deoxynucleotide and 2′-fluoro substituted mRNAs, which disrupt the hydrogen bonds between the A site codon and G530, A1492 and A1493. Our results show that multiple 2′-deoxynucleotide sub-stitutions in the mRNA substantially inhibit tRNA selection, whereas multiple 2′-fluoro substitutions in the mRNA have only modest effects on tRNA selection. Furthermore, the miscoding antibiotics paromomycin and streptomycin rescue the defects in tRNA selection with the multiple 2′-deoxynucleotide substituted mRNA. These results suggest that steric complementarity in the decoding center is more important than the hydrogen bonds between the A site codon and G530, A1492 and A1493 for tRNA selection.
© 2013 The Authors. Published by Elsevier Ltd.
Legend: The cover shows “shape recognition” spelled using the American Sign Language (http://www.asl.ms). Shape recognition (steric complementarity) in the decod-ing center of the ribosome is important for tRNA selection during protein synthesis.
0022-2836 © 2013 The Authors. Published by Elsevier Ltd.Open access underCC BY-NC-ND license. J. Mol. Biol. (2013) 425, 3778–3789
Introduction
During protein synthesis, the ribosome selects aminoacyl tRNAs corresponding to the codons in the mRNA. The aminoacyl tRNA binds as a ternary complex with elongation factor Tu (EF-Tu) and GTP to the aminoacyl site (A site) in the ribosome. The ribosome selects the cognate EF-Tu ternary complex with an error frequency of 1 × 10−3to 1 × 10−4.1,2 To achieve this level of accuracy in tRNA selection, the ribosome uses a kinetic proofreading mechanism consisting of two selection steps designated“initial selection” and “proofreading”, which are irreversibly separated by GTP hydrolysis on EF-Tu.3–6 More recent studies showed that the ribosome also uses an induced-fit mechanism to increase the accuracy of tRNA selection.7–9 These studies showed that the ribosome accelerates the rates of GTP hydrolysis and tRNA accommodation when a cognate EF-Tu ternary complex binds. In contrast, when a non-cognate or near-non-cognate EF-Tu ternary complex
binds to the ribosome, the rates of GTP hydrolysis and tRNA accommodation are inhibited. Thus, the accuracy of tRNA selection is greatly improved by accelerating two critical forward reactions (GTP hydrolysis and tRNA accommodation) when the cognate tRNA binds to the ribosome.
Insight into how the ribosome may use an induced-fit mechanism to accelerate the rates of GTP hydrolysis and tRNA accommodation was provided by X-ray crystallography.10 Crystal struc-tures showed that when a cognate tRNA binds to the A site, universally conserved 16S ribosomal RNA (rRNA) bases G530, A1492 and A1493 undergo a conformational change that stabilize their interac-tions with the minor groove of the codon–anticodon helix (Fig. 1a). Discrimination is achieved by G530, A1492 and A1493 precisely recognizing the Wat-son–Crick base pair geometry of the codon–antico-don helix by forming hydrogen bonds with the N3 of purine base, the O2 of pyrimidine base and the ribose 2′-hydroxyl groups (Fig. 1a).10 In addition,
(b)
(c)
(d)
A1493 A36 U1 G530 C518 Ser50 (S12) A1492 U2 A35 G530 C518 Pro48 (S12) U3 G34Position +4 Position +5 Position +6
(a)
Fig. 1. Interaction of the A site codon with the ribosome and equilibrium binding of EF-Tu ternary complex to the ribosome. (a) Interaction of the mRNA at positions + 4 to + 6 with the decoding center (Protein Data Bank ID: 1IBM). Shown are mRNA bases (U1, U2 and U3), tRNA bases (A36, A35 and G34), 16S rRNA bases (A1493, A1492, G530 and C518) and residues from ribosomal protein S12 (Ser50 and Pro48). Hydrogen bonds are represented by the dotted lines. (b–d) Representative equilibrium binding curves for EF-Tu (H84A)/Phe-tRNAPhe/GTP ternary complex binding to the ribosomal A site. Data are shown for the control mRNA (●) and mRNAs with a single 2′-deoxynucleotide substitution at positions +4 (■), + 5 (▲) and +6 (♦). The data were fit to a one-site binding hyperbolic equation to determine KD. (c) Representative binding curves for the control mRNA (●), mRNAs with two 2′-deoxynucleotide substitutions at positions +4 and +5 (■) or +5 and +6 (▲) and an mRNA with three 2′-deoxynucleotide substitutions at positions +4, +5 and +6 (♦). (d) Representative binding curves for the control mRNA (●), an mRNA with a single 2′-fluoro substitution at position +5 (■), an mRNA with two 2′-fluoro substitutions at positions + 5 and + 6 (▲) and an mRNA with three 2′-fluoro substitutions at positions +4, +5 and +6 (♦).
G530, A1492 and A1493 also interact by steric complementarity with the first and second base pairs of the codon–anticodon helix.10
Importantly, the binding of the cognate tRNA to the A site trigger a change in the structure of the 30S subunit from an open to a closed conformation (designated “domain closure”).11 Domain closure involves the rotation of the 30S subunit head toward the shoulder and the subunit interface and of the shoulder toward the inter-subunit space and the platform region.11Domain closure was proposed to accelerate GTP hydrolysis on EF-Tu and the accommodation of the cognate tRNA into the peptidyl transferase center.11,12By contrast, the anticodon of a non-cognate or near-cognate tRNA forms mis-matches with the codon, which distorts the geometry of the codon–anticodon helix and precludes stable interactions with G530, A1492 and A1493.11This in turn may inhibit domain closure and decrease the rates of GTP hydrolysis and tRNA accommodation.
However, recent crystal structures showed that G530, A1492 and A1493 form hydrogen bonds in a manner identical with that of cognate and near-cog-nate tRNAs in the A site.13,14 Furthermore, near-cognate tRNAs also induced domain closure to a similar extent as the cognate tRNA.13,14 Discrimina-tion against near-cognate tRNAs was proposed to occur because domain closure forced the codon– anticodon helix with unpaired bases at the first or second position to adopt the geometry of Watson– Crick base pairs.14 Since this is energetically unfavorable, the near-cognate tRNA dissociates from the ribosome.
To determine the contribution of the hydrogen bonds between the codon and G530, A1492 and A1493 in tRNA selection, we synthesized a library of mRNAs with single or multiple 2′-deoxynucleotide and 2′-fluoro substitutions in the A site codon, which systematically disrupt these hydrogen bonds. We used equilibrium binding and pre-steady-state kinet-ic methods to analyze the effects of the mRNA modifications on tRNA selection. Our results show that triple 2′-deoxynucleotide substitutions in the A site codon inhibits the rate of GTP hydrolysis by 30-fold and the rate of peptide bond formation by N4400-fold. In contrast, triple 2′-fluoro substitutions in the A site codon inhibit the rate of GTP hydrolysis by 12-fold and the rate of peptide bond formation by 4-fold only. The N1000-fold increase in the rate of peptide bond formation with the triple 2′-fluoro substituted codon compared to the triple 2 ′-deox-ynucleotide codon indicates that steric complemen-tarity is more important than the hydrogen bonds between the codon and G530, A1492 and A1493 for tRNA selection by the ribosome. Furthermore, we show that paromomycin and streptomycin can rescue the defects in peptide bond formation caused by the triple 2′-deoxynucleotide substitutions in the codon by 2700-fold. Since streptomycin induces
domain closure without stabilizing the interaction of A1492 and A1493 with the codon–anticodon helix,15
our results suggest that domain closure is important for tRNA accommodation on the ribosome.
Results
Binding of EF-Tu ternary complex to the ribosome is inhibited by multiple 2′-deoxynucleotide substitutions in the A site codon
To analyze the role of the hydrogen bonds formed by the 2′-hydroxyl groups in the A site codon in tRNA selection, we synthesized mRNAs with single, double or triple 2′-deoxynucleotide substitutions in the A site codon (positions + 4 to + 6). Previous studies have shown that 2′-deoxynucleotide sub-stitutions in the A site codon inhibit the binding of aminoacylated and deacylated tRNA to the ribosom-al A site.16–19However, these binding studies only examined the affinity of the tRNA in the fully accommodated A/A state on the ribosome. To analyze the effects of the 2′-deoxynucleotide sub-stitutions in the A site codon during initial selection, it is important to measure the binding affinity of the ternary complex in the A/T state on the ribosome. We, therefore, analyzed the binding of EF-Tu ternary complex in the A/T state to the ribosome. We used a mutant EF-Tu with the catalytic histidine 84 changed to alanine to inhibit GTP hydrolysis.20,21 Since EF-Tu (His84Ala) ternary complex cannot induce GTP hydrolysis in the time frame of the filter-binding assay, the ternary complex is in the A/T state on the ribosome. Ternary complex was formed with EF-Tu (His84Ala), GTP and 32P-labeled Phe-tRNAPheand filter-binding experiments were carried out with varying concentrations of ribosome complex. The equilibrium dissociation constant (KD) with the
unmodified control mRNA was 4.5 ± 0.3 nM, where-as the KDwith mRNAs having a single 2
′-deoxynu-cleotide substitution in the A site codon was increased by 1.6- to 4.2-fold (Table 1andFig. 1b).
Table 1. Binding and the rate of peptide bond for the different mRNAs mRNA KD(nM) kpep(s−1) Fp Control 4.5 ± 0.3 8.8 ± 1.4 0.94 ± 0.09 + 4D 7.3 ± 0.8 6.5 ± 1.4 0.92 ± 0.10 + 5D 18.9 ± 3.7 5.2 ± 1.0 0.85 ± 0.11 + 6D 12.9 ± 1.5 5.9 ± 0.1 0.86 ± 0.13 + 4D + 5D 274 ± 30 3.4 ± 0.3 0.51 ± 0.05 + 5D + 6D 278 ± 35 1.4 ± 0.2 0.35 ± 0.04 + 4D + 5D + 6D 445 ± 33 0.002 ± 0.001 b0.02 + 5F 5.8 ± 0.5 10.5 ± 0.2 0.96 ± 0.02 + 5F + 6F 14.4 ± 2.0 4.2 ± 0.6 0.97 ± 0.03 + 4F + 5F + 6F 11.0 ± 1.3 2.0 ± 0.1 0.55 ± 0.03 Fp, fraction of fMet-Phe dipeptide formed.
The KD with mRNAs having two and three 2
′-deoxynucleotide substitutions in the A site codon was increased by 63- and 110-fold, respectively (Fig. 1c). The binding data show that single 2′-deoxynucleotide substitutions in the A site codon only modestly inhibit the binding of EF-Tu ternary complex to the ribosome, whereas multiple 2 ′-deoxynucleotide substitutions in the A site codon are not thermodynamically additive but have a more drastic effect.
Multiple 2′-fluoro substitutions in the A site codon have small effects on EF-Tu ternary complex binding to the ribosome
The incorporation of a 2′-deoxynucleotide not only abolishes hydrogen bonding but also may disrupt the precise A-minor interactions in the decoding center because of the removal of the oxygen atom from the ribose sugar. Studies have shown that 2′-fluoro substitutions are less disruptive than 2′-deoxynucleotide substitutions in structured RNA.22 The 2′-fluoro substitution prefers the c3′-endo sugar pucker found in RNA23,24and the fluoro atom may participate as a weak hydrogen bond acceptor.22,25–30 In the decoding center, the 2
′-hydroxyl groups of the A site codon participate mainly as hydrogen bond donors (Fig. 1a). Therefore, the 2′-fluoro substitutions in the A site codon should disrupt the hydrogen bonds formed with G530, A1492 and A1493 without significantly interfering with steric complementarity.
To distinguish between these possibilities, we synthesized mRNAs with single (+ 5F), double (+ 5F + 6F) or triple (+ 4F + 5F + 6F) 2′-fluoro sub-stitutions in the A site codon. Filter-binding experiments showed that the KD with mRNA + 5F
was similar to the control mRNA (Table 1 and
Fig. 1d). The KDvalues with mRNAs having two or
three 2′-fluoro substitutions in the A site codon were increased by 2.5- to 3-fold compared to the control mRNA. Thus, ribosomes programmed with mRNAs having multiple 2′-fluoro substitutions in the A site codon showed dramatically improved binding affinity for EF-Tu ternary complex com-pared to mRNAs having multiple 2′-deoxynucleo-tide substitutions (Table 1). The rescue by the 2 ′-fluoro analogs indicate that the favorable steric complementarity of the A site codon is more important for binding EF-Tu ternary complex to the ribosome than the ability of the 2′-hydroxyl groups to form hydrogen bonds.
Fig. 2. Rate of GTP hydrolysis by EF-Tu ternary complex. (a) Representative time course for ribosome-dependent GTP hydroly-sis by EF-Tu ternary complex. Data are shown for the control mRNA (●) and mRNAs with a single 2′-deoxynucleotide substitution at positions + 4 (■), +5 (▲) and +6 (♦). The data were fit to a single exponential equation to obtain the rate of GTP hydrolysis. (b) Repre-sentative GTP hydrolysis data for the control mRNA (●), mRNAs with two 2′-deoxynucleotide substitu-tions at posisubstitu-tions + 4 and + 5 (■) or + 5 and + 6 (▲) and an mRNA with three 2′-deoxynucleotide substitu-tions at posisubstitu-tions + 4, + 5 and + 6 (♦). (c) Representative GTP hydro-lysis data for the control mRNA (●), an mRNA with a single 2′-fluoro substitution at position + 5 (■), an mRNA with two 2′-fluoro substitu-tions at posisubstitu-tions + 5 and + 6 (▲) and an mRNA with three 2′-fluoro sub-stitutions at positions + 4, + 5 and + 6 (♦). (d) Graph showing the rate of GTP hydrolysis at varying concen-tration of ribosomes for the control mRNA (●), an mRNA with two 2′-deoxynucleotide substitutions at positions +5 and +6 (■), an mRNA with three 2′-deoxynucleotide substitutions at positions +4, +5 and +6 (▲) and an mRNA with three 2′-fluoro substitutions at positions +4, +5 and + 6 (♦).
GTP hydrolysis by EF-Tu is inhibited by 2′-deoxynucleotide and 2′-fluoro substitutions in the A site codon
According to the kinetic model for tRNA selection, codon recognition by the cognate EF-Tu ternary complex triggers GTP hydrolysis on EF-Tu, whereas non-cognate ternary complex fails to trigger GTP hydrolysis and dissociate from the ribosome.7,9 To determine whether disrupting the interactions be-tween the codon and the ribosome affects GTP hydrolysis, we measured the pre-steady-state rate of GTP hydrolysis on EF-Tu with ribosomes having 2′-deoxynucleotide substitutions in the A site codon. GTP hydrolysis experiments were performed with a limiting concentration of EF-Tu ternary complex (0.1 μM final concentration) and a large excess of ribosomal complex (1.25 μM final concentration) to saturate the binding of the EF-Tu ternary complex to the ribosome (Fig. S1).31The rate of GTP hydrolysis was reduced by 2- to 4-fold with the single 2′-deoxynucleotide substituted mRNAs (Fig. 2a) (ko bs = 20.2 ± 1.2 s− 1, 10.0 ± 2.7 s− 1, 5.2 ±
0.9 s− 1 and 7.2 ± 1.5 s− 1 for control mRNA, mRNA + 4D, mRNA + 5D and mRNA + 6D, re-spectively). The rate of GTP hydrolysis was reduced by 16-, 22- and 50-fold with mRNA + 4D + 5D (kobs = 1.3 ± 0.1 s− 1), mRNA + 5D + 6D (kobs=
0 . 9 ± 0 . 3 s− 1) a n d m R N A + 4 D + 5 D + 6 D (kobs = 0.4 ± 0.2 s− 1), respectively (Fig. 2b and
Fig. S2). In contrast to the rate of GTP hydrolysis, the extent of GTP hydrolyzed by EF-Tu was similar in all cases. These results show that multiple 2 ′-deoxynu-cleotide substitutions in the A site codon inhibit GTP hydrolysis on EF-Tu possibly due to unfavorable interactions between the codon and the ribosome.
To find out whether the 2′-fluoro substitutions are better tolerated by the ribosome, we determined the rate of GTP hydrolysis on EF-Tu with ribo-somes programmed with mRNAs having the 2′-fluoro substitutions in the A site codon (Fig. S3). The rate of GTP hydrolysis was reduced by 3- to 7-fold with mRNA + 5F (kobs= 6.4 ± 1.9 s− 1),
mRNA + 5F + 6F (ko b s = 4.1 ± 0.3 s− 1) and
mRNA + 4F + 5F + 6F (kobs= 3.1 ± 0.6 s−1)
indi-cating that the 2′-fluoro substitutions are less disrup-tive than the 2′-deoxynucleotide substitutions in the A site codon (Fig. 2c). To estimate the saturation rate of GTP hydrolysis (kGTP), we measured the rate of GTP
hydrolysis at varying concentrations of ribosome complex and fit the data to a hyperbolic equation. Since large amounts of ribosomes, tRNAs, mRNAs and so on are required for this, we determined kGTP
only for the following representative mRNAs: control mRNA, mRNA + 5D + 6D, mRNA + 4D + 5D + 6D and mRNA + 4F + 5F + 6F (Fig. 2d). The kGTPwas
5 4 s− 1 f o r t h e c o n t r o l m R N A , 4 s− 1 f o r mRNA + 5D + 6D, 2 s−1for mRNA + 4D + 5D + 6D and 4 s−1 for mRNA + 4F + 5F + 6F. The kGTP is
substantially reduced with the modified mRNAs even at ribosome concentrations that are higher than the KD
for ternary complex binding to the ribosome. It is possible that the“induced fit” required for accelerating GTP hydrolysis on EF-Tu is compromised with these modified mRNAs bound to the ribosome.7–9
Multiple 2′-deoxynucleotide substitutions in the A site codon drastically inhibit peptide bond formation Following GTP hydrolysis on EF-Tu, the aminoa-cyl tRNA is accommodated into the 50S subunit A site and participates in peptide bond formation. To determine whether the hydrogen bonds formed by the 2′-hydroxyl groups in the A site codon with G530, A1492 and A1493 are important for tRNA accom-modation, we measured the rate of peptide bond formation with mRNAs having 2′-deoxynucleotide or 2′-fluoro substitutions (Fig. S4).31 Again, we used a limiting concentration of EF-Tu ternary complex (0.25μM final concentration) and a large excess of the ribosome complex (1.25μM final concentration) to overcome the binding defects observed with the 2′-deoxynucleotide substituted mRNAs. The maxi-mum extent of dipeptide formed was expected to be 20% because we used 5-fold lower concentration of EF-Tu ternary complex relative to the ribosome complex (see Materials and Methods). The rate of peptide bond formation with the control mRNA was 8.8 ± 1.4 s−1, which is similar to the saturation rate (kpep) reported previously at 20 °C.
9,32,33
The rates of peptide bond formation with single 2′-deoxynu-cleotide substituted mRNAs were similar to the control mRNA (Table 1 and Fig. 3a). In contrast, mRNAs with double 2′-deoxynucleotide substitu-tions showed a 2.6- to 6-fold reduced rate of peptide bond formation (Fig. 3b and Fig. S5). Additionally, the extent of dipeptide formed was reduced by 50– 65% compared to the control mRNA (Table 1). This indicated that, after GTP hydrolysis on EF-Tu,≈50% of the A site tRNA dissociated from the ribosome during the accommodation step. Finally, the extent of dipeptide formed was less than 2% in 10 s with the triple 2′-deoxynucleotide substituted mRNA (Fig. 3b). Therefore, we manually measured the rate of peptide bond formation for the triple 2 ′-deoxynucleotide substituted mRNA (Fig. S5e and f). Our conservative estimation is that mRNA with the triple 2′-deoxynucleotide substitutions is at least 4400-fold defective in the rate of peptide bond formation and is consistent with a previous report.32 Furthermore, the previous study showed that, with the triple 2′-deoxynucleotide substituted mRNA, the rate of peptide bond formation saturates at 0.5 μM ribosomes.32Thus, 2′-deoxynucleotide substitutions at all three positions in the A site codon drastically inhibited the accommodation of the tRNA into the A site in the 50S subunit and the tRNAs were largely rejected by the ribosome.
Multiple 2′-fluoro substitutions in the A site codon moderately inhibit peptide bond formation To determine whether the 2′-fluoro substitutions in the A site codon affect tRNA accommodation, we measured the rate of peptide bond formation with the 2′-fluoro substituted mRNAs (Fig. S6). The rate of peptide bond formation with a single 2′-fluoro substitution at position + 5 in the mRNA was similar to the control mRNA (Table 1 andFig. 3c). mRNA with the two 2′-fluoro substitutions at positions +5 and + 6 showed a 2-fold reduced rate of peptide bond formation (Fig. 3c). Surprisingly, the mRNA with the three 2′-fluoro substitutions at positions +4, + 5 and + 6 showed only a 4-fold reduced rate of peptide bond formation and the extent of dipeptide formed was reduced by 45% compared to the control mRNA (Fig. 3c). This is at least a 1000-fold increase in the rate of peptide bond formation relative to the mRNA with the three 2′-deoxynucleotide substitu-tions in the A site codon (Fig. 3d). This remarkable recovery both in the rate of peptide bond formation and in the extent of dipeptides formed with mRNA + 4F + 5F + 6F indicate that steric comple-mentarity of G530, A1492 and A1493 with the codon–anticodon helix are critical for the
accommo-dation of the tRNA into the A site and for peptide bond formation.
Paromomycin and streptomycin restore the rates of GTP hydrolysis by EF-Tu and peptide bond formation
We wondered to what extent could miscoding antibiotics compensate for the loss of favorable interactions with the 2′-deoxynucleotide substituted A site codons. Antibiotics such as paromomycin, neomycin and streptomycin are known to reduce the fidelity of translation.34 In addition, some of these miscoding antibiotics have been shown to stimulate the translation of single-stranded DNA,35,36 poten-tially by stabilizing codon–anticodon interactions in the A site. Paromomycin binds to helix 44 in the decoding center and displaces A1492 and A1493 toward the codon–anticodon helix in the A site.10,15
Paromomycin, therefore, makes the interaction of A1492 and A1493 with the codon–anticodon helix of the near-cognate tRNA energetically more favorable and also induces domain closure explaining the reduced fidelity of translation.10Consistent with this proposal, kinetic studies showed that paromomycin accelerates the rates of GTP hydrolysis and
Fig. 3. Rate of peptide bond formation. (a) Representative time course showing the kinetics of dipeptide formation. Ribosomes contained f[35S]Met-tRNAfMet in the P site and were reacted with EF-Tu/Phe-tRNAPhe/GTP ternary complex. Data are shown for the control mRNA (●) and mRNAs with a single 2′-deoxynucleotide substi-tution at positions + 4 (■), +5 (▲) and + 6 (♦). The data were fit to a single exponential equation to obtain the rate of peptide bond formation. (b) Representative peptide bond for-mation data for the control mRNA (●), mRNAs with two 2′-deoxynu-cleotide substitutions at positions + 4 and + 5 (■) or +5 and +6 (▲) and an mRNA with three 2′-deoxynu-cleotide substitutions at positions + 4, + 5 and + 6 (♦). (c) Representa-tive peptide bond formation data for the control mRNA (●), an mRNA with a single 2′-fluoro substitution at position + 5 (■), an mRNA with two 2′-fluoro substitutions at positions + 5 and + 6 (▲) and an mRNA with three 2′-fluoro substitutions at posi-tions + 4, + 5 and + 6 (♦). (d) Bar graph showing the rate of peptide bond formation with the 2′-deoxynucleotide and the 2′-fluoro substituted mRNAs. Error bars represent SD from two experiments.
accommodation of near-cognate tRNAs.37 We determined the rate of GTP hydrolysis in the presence of paromomycin with the control mRNA and the triple 2′-deoxynucleotide substituted mRNA (Fig. 4a and Fig. S7). In the presence of paromo-mycin, the rate of GTP hydrolysis with the triple 2 ′-deoxynucleotide substituted mRNA (kobs= 10.2 ±
3 s−1) was similar to the control mRNA (kobs=
11.5 ± 1 s−1). We also tested streptomycin, another miscoding antibiotic that binds to a site that is distinct from the paromomycin-binding site on the ribosome.15 Streptomycin is proposed to reduce the fidelity of translation by inducing “domain closure” with near-cognate tRNAs.15 , 38 We measured the rate of GTP hydrolysis in the presence of streptomycin (Fig. S8). Streptomycin also increased the rate of GTP hydrolysis with the triple 2′-deoxynucleotide substituted mRNA (kobs= 4.9 ±
1 s−1) to almost the same rate as with the control mRNA (kobs= 7.3 ± 0.1 s−1) (Fig. 4a). We next
tested the rate of peptide bond formation in the presence of paromomycin and streptomycin (Figs. S9 and S10). Both miscoding antibiotics substan-tially improved the rate and the extent of peptide formation with the triple 2′-deoxynucleotide and the triple 2′-fluoro substituted mRNAs (Table 2 and
Fig. 4b). These results showed that paromomycin and streptomycin can compensate for the unfavor-able interactions that the triple 2′-deoxynucleotide and 2′-fluoro substituted mRNAs make with the decoding center.
Fidelity of protein synthesis is decreased by 2′-deoxynucleotide substitutions in the A site codon
To determine whether the loss of the hydrogen bonds and steric complementarity between the A site codon and the ribosome lowers the fidelity of protein synthesis, we measured the error frequency of translation. Ribosomes programmed with the different mRNAs and with f[35S]Met-tRNAfMetin the P site were reacted with a mixture of aminoacylated tRNAs (Fig. S11).31 The error frequency was calculated from the ratio of fMet-Leu (incorrect dipeptide) to Phe (correct dipeptide) + fMet-Leu (incorrect dipeptide) formed. The error frequen-cy with mRNA + 4D, mRNA + 5F, mRNA + 5F + 6F and mRNA + 4F + 5F + 6F were similar to the control mRNA (Fig. 4c). mRNA + 5D and mRNA + 6D showed about a 2-fold higher error
Fig. 4. Effect of paromomycin and streptomycin on the rates of GTP hydrolysis and peptide bond formation. (a) Bar graph showing the rates of GTP hydrolysis by EF-Tu ternary complex with the control mRNA and with the mRNA having three 2′-deoxynucleotide substitutions at positions +4, +5 and +6. Experiments were performed in the absence of antibiotics (white bar), in the presence of paromomycin (gray bar) or in the presence of streptomycin (black bar). (b) Bar graph showing the rates of peptide bond formation with the control mRNA, with the mRNA having three 2′-deoxynucleotide substitutions at positions + 4, + 5 and + 6 and with the mRNA having three 2′-fluoro substitutions at positions +4, +5 and +6. Labels for the bar graph are as indicated above. The asterisks are used to indicate that, in the absence of antibiotics, the rate of peptide bond formation was very slow with the triple 2′-deoxynucleotide substituted mRNA. (c) Bar graph showing the error frequency with 2′-deoxynucleotide and 2′-fluoro substituted mRNAs. The asterisks are used to indicate that only negligible amounts of dipeptides were formed with the triple 2′-deoxynucleotide substituted mRNA. In all cases, the error bars represent SD from at least two experiments.
Table 2. Rate of peptide bond formation with antibiotics
mRNA kpep(s−1) Fp Control (Par) 11.4 ± 0.7 0.85 ± 0.13 Control (Str) 9.0 ± 0.6 0.95 ± 0.28 + 4D + 5D + 6D (Par) 7.2 ± 0.3 0.84 ± 0.01 + 4D + 5D + 6D (Str) 5.4 ± 0.9 0.78 ± 0.03 + 4F + 5F + 6F (Par) 9.9 ± 0.6 0.95 ± 0.09 + 4F + 5F + 6F (Str) 7.7 ± 0.7 1.07 ± 0.03 Fp, fraction of fMet-Phe dipeptide formed; Par, paromomycin; Str,
frequency. Interestingly, mRNA + 4D + 5D and mRNA + 5D + 6D showed 3- and 7-fold higher error frequency, respectively, than the control mRNA. The increase in the error frequency with mRNA + 4D + 5D and mRNA + 5D + 6D is due to the reduced formation of the correct fMet-Phe dipeptide. We could not determine error frequency with mRNA + 4D + 5D + 6D because the amount of correct fMet-Phe dipeptide formed was negligible. These results show that disrupting the interactions between the codon and the A site lowers the fidelity of protein synthesis by decreasing the efficiency with which the ribosome accepts the cognate tRNA.
Discussion
The mechanism of tRNA selection by the ribosome has been investigated for more than 30 years. Recent X-ray crystal structures provide a structural explanation for how the ribosome is able to discriminate between a cognate tRNA and a near-cognate tRNA. Crystal structures showed that the ribosome recognizes the geometry of the cognate codon–anticodon helix by using universally conserved bases G530, A1492 and A1493 in the 16S rRNA (Fig. 1a).10,39 The interaction of G530, A1492 and A1493 with the cognate codon –antico-don helix induces domain closure, GTP hydrolysis by EF-Tu and the accommodation of the cognate tRNA into the ribosome. In contrast, G530, A1492 and A1493 fail to interact stably with near-cognate codon–anticodon helix and fail to induce domain closure causing the near-cognate tRNA to dissociate from the ribosome.11,40 However, more recent crystal structures show that G530, A1492 and A1493 interact with the cognate and near-cognate codon–anticodon helix in an identical manner.13,14
In addition, domain closure is induced even when a near-cognate tRNA binds to the A site, which forces the bases in the codon–anticodon helix to adopt the geometry of Watson–Crick base pairs. Since this is energetically unfavorable, it causes the near-cog-nate tRNA to dissociate from the ribosome. Thus, there are now two different structural models that explain the mechanism of tRNA selection by the ribosome.
To understand the mechanism of tRNA selection, we focused on the interactions between the 2 ′-hy-droxyl groups of the A site codon and bases G530, A1492 and A1493. Previous studies showed that single 2′-deoxynucleotide substitutions in A site codon inhibited the binding of tRNA to the A site by 3- to 4-fold,17,18 while 2′-deoxynucleotide substitu-tion at all three posisubstitu-tions in the A site codon reduced the tRNA binding affinity by 10-fold.16 Since the A site tRNA does not directly contact the 2′-hydroxyl groups in the codon, these results are consistent with the idea that the loss of the 2′-hydroxyl groups
from the A site codon inhibits“domain closure” and weakens the interaction of the tRNA with the ribosomal A site.17However, no studies have been carried out to systematically examine the role of these 2′-hydroxyl groups in the A site codon in individual steps of the tRNA selection pathway.
The first step in the tRNA selection pathway is the interaction of the EF-Tu ternary complex with the ribosome. We determined the binding affinity of EF-Tu ternary complex in the A/T state on the ribosome. Our studies showed that single 2′-deoxynucleotide substitutions in the A site codon inhibit the binding of EF-Tu ternary complex modestly, whereas double or triple 2′-deoxynucleotide substitutions drastically inhibit the binding of EF-Tu ternary complex to the ribosome (KD is 100-fold higher with the triple
2′-deoxynucleotide substituted mRNA compared to the control mRNA). Therefore, the tRNA bound to EF-Tu⋅GTP, in the A/T state on the ribosome, is more sensitive to 2′-deoxynucleotide substitutions in the A site codon compared to the previous tRNA binding studies, which analyzed the fully accommodated A/A state.16–18A possible explanation for this difference is that the tRNA bound to EF-Tu⋅GTP in the A/T state on the ribosome is in a strained conformation compared to the fully accommodated tRNA in the A/A state (see below).41 Interestingly, the KD of
EF-Tu ternary complex binding to the ribosome is increased by only 2-fold with the triple 2′-fluoro substituted A site codon. This suggests that steric complementarity in the decoding center dominates the interaction of the EF-Tu ternary complex with the ribosome and is consistent with the hypothesis proposed by Potapov.42
We next analyzed the importance of the 2 ′-hydroxyl groups in the A site codon for two key steps in tRNA selection: GTPase activation on EF-Tu and tRNA accommodation. Since GTPase activation is rate limiting for GTP hydrolysis, and tRNA accommodation is rate limiting for peptide bond formation, we measured these chemical steps using well-established pre-steady-state kinetic assays.7,9 Our studies showed that single 2′-deox-ynucleotide or 2′-fluoro substitutions in the A site codon only modestly inhibit the rates of GTP hydrolysis and peptide bond formation. However, triple 2′-deoxynucleotide substitutions in the A site codon inhibited the rates of GTP hydrolysis and peptide bond formation by 30- and N4400-fold, respectively. In contrast, triple 2′-fluoro substitutions in the A site codon inhibited the rates of GTP hydrolysis and peptide bond formation by 12- and 4-fold, respectively. The significant recovery with the triple 2′-fluoro substituted A site codon suggests that the hydrogen bonds formed by the 2′-hydroxyl groups in the A site codon with G530, A1492 and A1493 are not the major determinant for GTP hydrolysis and peptide bond formation. Rather, tRNA selection mainly depends on G530, A1492
and A1493 recognizing the geometry of the codon– anticodon helix by steric complementarity (using favorable van der Waals interaction, hydrophobic contacts and base stacking). Indeed, previous studies have shown that the A-minor interactions critically depend on steric complementarity between adenine and the minor groove, which provide optimal van der Waals contact, hydrogen bonding and a hydrophobic environment creating a highly energetically favorable interaction.43–47
Interestingly, the extent of GTP hydrolyzed is similar with all the mRNAs tested, whereas the extent of dipeptide formed is severely reduced especially with the triple 2′-deoxynucleotide substituted A site codon (b2% fMet-Phe dipeptide formed) (Fig. 3b). This shows that the aminoacyl tRNA is rejected after GTP hydrolysis during the accommodation of the tRNA. Surprisingly, in the presence of paromomycin or streptomycin, both the extent and the rates of GTP hydrolysis and peptide bond formation were similar with the control mRNA and the triple 2′-deoxynucleotide substituted mRNA (Table 2). The complete recovery in the extent of fMet-Phe formed and the≈3000-fold improvement in the rate of peptide bond formation with the triple 2′-deoxynucleotide substituted mRNA in the pres-ence of paromomycin or streptomycin provide new insights into the mechanism of tRNA selection. Paromomycin stabilizes the flipped out conformation of A1492 and A1493.10However, streptomycin does not stabilize the flipped out conformation of A1492 and A1493.15On the other hand, both paromomycin (with a tRNA in the A site) and streptomycin (even without a tRNA in the A site) induce domain closure.10,11,15 Since streptomycin in effect uncou-ples codon–anticodon recognition from domain closure, it suggests that the improvement in the rates of GTP hydrolysis and peptide bond formation with the miscoding antibiotics are due to the stabilization of the domain closed state of the ribosome. Thus, domain closure is critical for preventing the rejection of the tRNA with the triple 2′-deoxynucleotide substituted A site codon during the proofreading step.
Our results show that steric complementarity by G530, A1492 and A1493 with the cognate codon– anticodon complex and domain closure play an active role in tRNA selection and are consistent with the model proposed by Ogle et al.10 and Ogle et al.11 We speculate that the increased rate of rejection for the near-cognate ternary complex during proofreading is largely because of the failure to induce domain closure. Both cognate and near-cognate tRNAs are in a strained configuration when bound to EF-Tu⋅GTP on the ribosome (in the A/T state).40,41,48–50 Nonetheless, domain closure occurs only with the cognate tRNA and it causes a tightening of the decoding center around the antico-don arm of the tRNA.11,40GTP hydrolysis on EF-Tu
and the dissociation of EF-Tu/GDP from the ribo-some allows the acceptor arm of the cognate tRNA to swing into the A site on the 50S subunit, which relaxes the tRNA into its canonical structure.48 In contrast, due to the lack of steric complementarity in the decoding center and the absence of domain closure, the interaction of the near-cognate tRNA with the 30S subunit is weakened. This may allow the near-cognate tRNA to relax from the A/T state without coupling it to the movement of the acceptor arm into the A site on the 50S subunit resulting in the rejection of the near-cognate tRNA from the ribosome.
Materials and Methods
Preparation of ribosomes, mRNAs, tRNAs and EF-Tu Tight-couple ribosomes were purified from Escherichia coli MRE600 and washed in high-salt buffer as previously described.31 We purchased, from Dharmacon, synthetic mRNAs with the following sequence: 5′-AAGGAG-GUAAAAAUGUUUGCU-3′, where the underlined nucleo-tides correspond to the A site codon (positions + 4 to + 6). 2′-Deoxynucleotide or 2′-fluoro substitutions were incor-porated during synthesis at positions + 4 to + 6 in the mRNAs. E. coli tRNAPhe was purified as described previously.31 EF-Tu, EF-Tu (H84A) and nucleotide-free EF-Tu were purified using the IMPACT-CN system according to the supplier's protocol (New England Biolabs) and as previously described.31Aminoacylation of tRNAfMet and tRNAPhe were performed using purified E. coli histidine-tagged synthetase, essentially as previously described.51 Formylation of initiator tRNAfMet was per-formed as previously described.51 The aminoacylated tRNAs were purified by HPLC on a C18 reverse phase column.51 The extent of aminoacylation was verified by acid gel electrophoresis, and the level of aminoacylation was greater than 95%. f[35S]Met-tRNAfMet was prepared as previously described.52
Equilibrium binding of EF-Tu ternary complex to the ribosomal A site
E. coli tRNAPhe was 32P-labeled at the 3′-end using [α-32
P]ATP and tRNA nucleotidyl transferase as previ-ously described.53 Equilibrium binding of EF-Tu ternary complex to the A site was determined as described previously.54 Filter-binding experiments were performed in buffer A [50 mM Tris–HCl (pH 7.5), 70 mM NH4Cl, 30 mM KCl, 15 mM MgCl2, 0.5 mM spermidine, 8 mM putrescine and 2 mM DTT] so that the endpoints for binding reached 70–80% with the 2′-deoxynucleotide substituted mRNAs.55Briefly, initiation complexes were prepared by incubating activated 70S ribosomes, mRNA (10-fold excess over ribosome) and tRNAfMet(5-fold excess over ribosome) at 37 °C for 30 min. The initiation complex was diluted to give a range of concentrations (0.1–1000 nM as shown in Fig. 1) using buffer A. We transferred 15μl of each initiation complex dilution to a 96-well conical bottom plate (Nunc). EF-Tu (H84A) ternary complex was prepared by combining EF-Tu (H84A) (2 nM), GTP (1 mM),
phosphoenol pyruvate (3 mM) and pyruvate kinase (0.25μg/μl) in buffer A and incubating at 37 °C for 30 min and then mixed with 3′-32
P-labeled Phe-tRNAPhe(0.2 nM). The ternary complex reaction mix was incubated at 37 °C for 5 min and then placed on ice. We added 15μl of ternary complex to the initiation complexes present in the 96-well plate (final volume, 30μl), mixed and incubated at room temperature for 1 min. We filtered 25μl of the final reaction mix through a modified 96-well filtration apparatus having a nitrocellulose membrane (0.45μm; Osmonics) at the top and a nylon membrane (0.45μm; Amersham) at the bottom.56The filters were washed three times with 100μl
of buffer A per well. The membranes were dried and quantified with a phosphorimager (Bio-Rad). All binding experiments were independently repeated more than three times. The equilibrium dissociation constant (KD) was determined by fitting the binding data to a one-site binding hyperbolic equation (GraphPad Prism).
Kinetics of GTP hydrolysis by EF-Tu ternary complex GTP hydrolysis experiments were performed in high-fidelity buffer B [50 mM Tris–HCl (pH 7.5), 70 mM NH4Cl, 30 mM KCl, 3.5 mM MgCl2, 0.5 mM spermine, 8 mM putrescine and 2 mM DTT], essentially as described previously.31In a typical experiment, initiation complexes were prepared in buffer B by incubating activated 70S ribosomes (2.5μM), mRNA (5 μM) and tRNAfMet (3.75μM) at 37 °C for 10 min. Ternary complex was prepared by mixing nucleotide-free EF-Tu (1μM), [γ-32P] GTP (0.2μM) and HPLC-purified Phe-tRNAPhe(1μM) and incubating at 37 °C for 5 min. To determine the rate of GTP hydrolysis, we rapidly mixed 15μl of 70S initiation complex (2.5μM) with 15 μl of EF-Tu/GTP/Phe-tRNAPhe ternary complex (0.20μM) and quenched with 15 μl of 40% formic acid in a quench-flow instrument (μQFM-400; BioLogic). The concentration of ribosome and ternary complexes was 1.25μM and 100 nM, respectively, after mixing. To determine the saturation rate of GTP hydrolysis, we varied the concentration of the ribosome as indicated in the figure. Experiments with streptomycin (Sigma) and par-omomycin (Sigma) were performed by adding 400μM antibiotics to the initiation complex and incubating for 10 min at room temperature before performing quench-flow experiments. Free phosphate was separated by polyethyleneimine-cellulose TLC in 0.5 M potassium phosphate (pH 3.5). The extent of hydrolysis was quanti-fied with a phosphorimager (Bio-Rad). All experiments were repeated independently at least two times. The GTP hydrolysis rate was determined by fitting the data to a single exponential equation (GraphPad Prism).
Rate of peptide bond formation
The 70S initiation complex and the EF-Tu/GTP/Phe-tRNAPhe ternary complex were prepared as described previously.31To determine the rate of dipeptide formation, we rapidly mixed 15μl of 70S initiation complex (2.5 μM) with 15μl of the reaction mix containing EF-Tu/GTP/Phe-tRNAPhe(0.5μM) and quenched it with 15 μl of 1 M KOH in a quench-flow instrument (μQFM-400; BioLogic). In the case of the triple 2′-deoxynucleotide substituted mRNA, the reaction was very slow and the rate of dipeptide
formation was determined manually. Experiments with streptomycin and paromomycin were performed by adding 400μM antibiotics to the initiation complex and incubating for 10 min at room temperature before loading on to the quench-flow instrument. The dipeptide was resolved by electrophoresis on cellulose TLC plates and quantified using a phosphorimager (Bio-Rad). All experiments were repeated independently at least two times. The rate of peptide bond formation was determined by fitting the data to a single exponential equation (GraphPad Prism). Because we used 70S complex having f[35S]Met-tRNAfMet in the P site at 5-fold excess over the EF-Tu/GTP/Phe-tRNAPheternary complex, we expect the maximum extent of dipeptide formed to be 20%. Therefore, the fraction dipeptide formed: (Fp) = %dipeptide formed/20.
Determination of error frequency
Fidelity experiments were performed as described earlier.31The dipeptides f[35S]Met-Phe and f[35S]Met-Leu were resolved by electrophoresis on cellulose TLC plates and quantified using a phosphorimager (Bio-Rad). The extent of misincorporation was estimated by calculating the ratio of f[35S]Met-Leu/f[35S]Met-Phe + f[35S]Met-Leu.
Acknowledgements
We thank Jack Kyte and Dan Herschlag for useful discussions and Ulrich Muller for comments on the manuscript. This work was supported by National Institutes of Health Grant GM065265 to S.J.
Supplementary Data
Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.jmb.2013.02.038 Received 18 December 2012; Received in revised form 23 February 2013; Accepted 27 February 2013 Available online 27 March 2013 Keywords: protein synthesis; elongation factor Tu; kinetics; peptide bond; GTP hydrolysis Abbreviations used:
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