Thio-Modification of tRNA at the Wobble Position as Regulator of the

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Thio-Modification of tRNA at the Wobble Position as Regulator of the Kinetics of Decoding and Translocation on the Ribosome Namit Ranjan and Marina V. Rodnina* Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Goettingen, Germany S Supporting Information *

ABSTRACT: Uridine 34 (U34) at the wobble position of the tRNA anticodon is post-transcriptionally modified, usually to mcm5s2, mcm5, or mnm5. The lack of the mcm5 or s2 modification at U34 of tRNALys, tRNAGlu, and tRNAGln causes ribosome pausing at the respective codons in yeast. The pauses occur during the elongation step, but the mechanism that triggers ribosome pausing is not known. Here, we show how the s2 modification in yeast tRNALys affects mRNA decoding and tRNA−mRNA translocation. Using real-time kinetic analysis we show that mcm5-modified tRNALys lacking the s2 group has a lower affinity of binding to the cognate codon and is more efficiently rejected than the fully modified tRNALys. The lack of the s2 modification also slows down the rearrangements in the ribosome−EF-Tu−GDP−Pi−Lys-tRNALys complex following GTP hydrolysis by EF-Tu. Finally, tRNA−mRNA translocation is slower with the s2-deficient tRNALys. These observations explain the observed ribosome pausing at AAA codons during translation and demonstrate how the s2 modification helps to ensure the optimal translation rates that maintain proteome homeostasis of the cell.

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INTRODUCTION Transfer RNAs (tRNAs) are post-transcriptionally modified in all living organisms. Currently, more than 100 different modifications are known, some common to all tRNAs, while others specific for only a few tRNA species. In particular position 34 in the tRNA anticodon, which interacts with the third (wobble) position of the codon, has emerged as a hotspot for modifications.1,2 Modified uridine 34 (U34) is usually found in tRNAs that read split codon-box families where the synonymous A- and G-ending codons encode a different amino acid than the U- and C-ending codons. U34 is posttranscriptionally modified in all domains of life. In yeast, U34 is modified in 11 out of 42 tRNA species to 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-carbamoylmethyluridine (ncm5U) or 5carbamoylmethyl-2′-O-methyluridine (ncm5Um).1 The modifications ensure correct tRNA folding,3 stabilize the conformation of the tRNA anticodon loop,4,5 facilitate the interactions with aminoacyl-tRNA (aa-tRNA) synthetases,6 modulate decoding and ribosome processivity,7 and assist in the noncanonical function of priming reverse transcription of human immunodeficiency virus type-1 (HIV-1).8 In yeast and higher eukaryotes, U34 in tRNALys (anticodon UUU), tRNAGln (anticodon UUG) and tRNAGlu (anticodon UUC) carries the mcm5s2 modification, which is introduced by the enzymes of the ELP and URM1 pathways. The enzymes of the ELP pathway include the six subunits of the Elp-complex (Elp1p-6p) and the tRNA methyltransferase complex (Trm9p and Trm112p).9,10 Thiolation (s2) is achieved by URM1 pathway by the action of the ubiquitin-related modifier 1 © 2017 American Chemical Society

(Urm1p), its activating enzyme Uba4p, thiouridine modification protein 1 (Tum1p), and proteins called “Needs Cla4 to Survive 2 and 6” (Ncs2p and Ncs6p). Uba4p first activates URM1 as acyl-adenylate and then transfers the sulfur on the catalytic cysteine in Urm1p. Subsequently, Ncs2p/Ncs6p mediate the transfer of sulfur from Urm1p to tRNA in an ATP-dependent manner.11,12 In yeast, single tRNA mutants lacking the mcm5 or s2 modification show numerous phenotypes, including temperature and stress sensitivity.11,13 Elevated temperatures and other stresses reduce the levels of s2 modification.14,15 In higher eukaryotes, hypomodification is associated with neurological disorders, type 2 diabetes, cancer and mitochondrial-linked disorders in humans,16−18 neurological and developmental dysfunctions in Caenorhabditis elegans,19 and perturbations in plant immunity.20 These phenotypes arise from the altered proteome composition of the cell due to specific changes at the level of translation. Deletions of URM1 or ELP3, the genes that are responsible for the s2 and mcm5 modifications of U34 in tRNALys, tRNAGln and tRNAGlu in yeast impede translation of mRNAs with repeats of AAA, CAA and GAA codons read by these tRNAs, whereas the global protein synthesis is hardly affected.21 The lack of the s2 modification results in a higher ribosome density at these codons in ribosome profiling experiments, also suggesting a direct involvement of the modification at some step of translation elongation.22,23 Ribosome pausing can arise at any stage of translation Received: January 27, 2017 Published: April 3, 2017 5857

DOI: 10.1021/jacs.7b00727 J. Am. Chem. Soc. 2017, 139, 5857−5864

Article

Journal of the American Chemical Society

Figure 1. Kinetics of A-site binding. (a) Kinetic scheme of A-site binding.34 (b) Fluorescence changes of [14C]Lys-tRNALys(Prf16/17) reporting initial binding. The ternary complex of EF-Tu−GTP with [14C]Lys-tRNALys(Prf16/17) containing the mcm5s2U34 (black) or the mcm5U34 (blue) modification (0.1 μM, all concentrations after mixing) was rapidly mixed with nonprogrammed ribosomes (2.0 μM) in a stopped-flow apparatus, and fluorescence changes of Prf were monitored. Here and in other panels of Figure 1, the fluorescence traces were normalized to start at 1 to show relative fluorescence and are shifted relative to one another for better visualization. Smooth lines represent fits obtained by global evaluation of the combined time courses obtained at various concentrations using numerical integration (Materials and Methods). Two control time courses showing no fluorescence change were obtained by mixing ternary complex with buffer only. For original data, see Figure S1 and S2. Each trace is an average of 6 individual time courses with mcm5s2U34 tRNALys (n = 6) or 8 individual time courses with s2U34 tRNALys (n = 8). a.u., arbitrary units. (c) Conformational changes of [14C]Lys-tRNALys(Prf16/17) upon decoding of the cognate AAA codon. The experiment was carried out as in (b), except that programmed ribosome complexes were used with fMet-tRNAfMet bound at the AUG codon in the P site and an AAA codon exposed in the A site (2.0 μM; n = 5). (d) Dissociation from the codon-recognition complex. The ternary complexes of EF-Tu(H84A)−GTP with [14C]LystRNALys(Prf16/17) containing the mcm5s2U34 modification (black) or the mcm5U34 modification (blue) was chased with 10-fold excess ternary complex with nonfluorescent [14C]Lys-tRNALys (n = 5). (e) Affinity of the codon recognition complex. Equilibrium binding of the ternary complex EF-Tu(H84A)−GTP−[14C]Lys-tRNALys(Prf16/17) (40 nM) to programmed ribosomes (added up to 350 nM) was monitored in a spectrofluorimeter (Materials and Methods). Continuous red lines represent fits obtained using a quadratic equation; the data were normalized by setting the first value (no ribosomes added) to 0 and the saturation value to 1. Data were obtained in 3 independent experiments. (f) Conformational changes of EF-Tu monitored by mant-GTP/mant-GDP fluorescence. Ternary complex of EF-Tu−mant-GTP with [14C]LystRNALys with mcm5s2U34 (black) or mcm5U34 modification (blue) (0.1 μM) was rapidly mixed with programmed ribosomes (2.0 μM) in a stoppedflow apparatus, and fluorescence changes of mant were monitored (n = 5). (g) GTP hydrolysis. Ternary complex EF-Tu−[γ-32P]GTP−[14C]LystRNALys with mcm5s2U34 (black symbols) or mcm5U34 (blue symbols) modification (0.1 μM) was rapidly mixed with programmed ribosomes (2.0 μM) in a quench-flow apparatus; the extent of GTP hydrolysis was analyzed by TLC (Materials and Methods). (h) Pi release. Ternary complex EFTu−GTP−[14C]Lys-tRNALys with mcm5s2U34 (black) or mcm5U34 (blue) modification (0.1 μM) was rapidly mixed with programmed ribosomes (2.0 μM) in a stopped-flow apparatus in the presence of MDCC-PBP (2.5 μM). Pi released from EF-Tu is captured by MDCC-PBP resulting in a fluorescence change (n = 7). (i) Peptide bond formation was monitored upon rapidly mixing ternary complexes EF-Tu−GTP−[14C]Lys-tRNALys with mcm5s2U34 (black) and mcm5U34 (blue) (0.1 μM) with initiation complex (2.0 μM) in a quench-flow apparatus, and the extent of peptide formation was analyzed by HPLC and radioactivity counting. 5858

DOI: 10.1021/jacs.7b00727 J. Am. Chem. Soc. 2017, 139, 5857−5864

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Journal of the American Chemical Society

Figure 2. Rate constants of A-site binding for mcm5s2 or mcm5-modified Lys-tRNALys. (a) Kinetic model used for numerical integration. (b) Comparison of the elemental rate constants from global fitting. Error bars are SEM of the fit; see also Figures S1 and S2 and Table S1.

G-promoted tRNA−mRNA translocation is impeded when the modification is lacking. Our findings provide an insight into the contribution of modifications at the tRNA wobble position to translation and suggest how the lack of a single modification can lead to ribosome pausing during translation.

elongation, e.g., mRNA decoding, peptide bond formation, and tRNA−mRNA translocation. Pausing leads to defective cotranslational folding of nascent peptides22 and frameshifting.24 On the basis of the crystal structure of tRNALys from Escherichia coli, which carries the mcm5s2 U34 modification and can read both Lys codons AAA and AAG, the s2 group is involved in stacking with U35, which stabilizes a particular conformation of the anticodon loop.25 In E. coli tRNALys, mcm5s2 U34 at the first anticodon position is involved in a dual mode of base-pairing interactions with the third codon position A and G. While the effect of the modification on reading the AAG codon appears to be through allowing a zwitterionic form of the base,25 it is unclear what effect the sulfur would have for a normal A-U base pair. Unlike E. coli, yeast cells have two tRNALys isoacceptors, the mcm5s2-modified tRNALys (anticodon UUU) complementary to the AAA codon, and another tRNALys with the anticodon CUU complementary to the AAG codon. Our initial results suggested that the lack of the s2 modification in tRNALys(UUU) reduces the rate of Lys incorporation and the stability of peptidyl-tRNALys binding to the ribosome.21 How the lack of modification leads to ribosome stalling at the AAA codons remained unclear. Here, we analyze the contribution of the s2U34 modification on the function of tRNALys during translation elongation in vitro using ensemble stopped-flow and quench-flow kinetic approaches. We identify the decoding steps that are affected by the lack of s2U34 modification in Lys-tRNALys, including the dissociation steps that result in aggressive proofreading of the hypomodified Lys-tRNALys on the cognate codon. Surprisingly, the lack of s2 modification also affects the post-GTP-hydrolysis rearrangements of the EF-Tu−ribosome complex, resulting in a delay in Lys-tRNALys delivery into the A site. Furthermore, EF-

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RESULTS The Effect of U34 Thiolation on Decoding. The kinetic analysis of decoding was carried out using the established kinetic model of A-site binding (Figure 1a).26 In the first step, the ternary complex consisting of EF-Tu, GTP, and aa-tRNA binds to the ribosome to form a labile initial binding complex with rate constants k1 and k−1 for the forward and backward reactions, respectively. Recruitment of the ternary complex− presumably through interactions between the GTP-binding domain of EF-Tu and ribosomal protein bL1227,28 − allows for codon scanning by the anticodon of aa-tRNA.29 Codon reading is rapid and reversible and precedes the formation of the stable codon-anticodon complex.30 Because the codon-reading step is not resolved by ensemble kinetics, we grouped codon reading and initial binding for the kinetic analysis. Codon recognition (rate constants k2 and k−2), which likely also includes the domain closure of the small ribosomal subunit (SSU),31,32 triggers the GTPase activation of EF-Tu (k3) and GTP hydrolysis (kGTP) on the large ribosomal subunit (LSU).33 As the steps of GTPase activation and GTP hydrolysis are not resolved kinetically,33,34 these two steps were grouped as well, and the reaction was considered irreversible due to irreversible GTP cleavage. The subsequent release of inorganic phosphate (Pi) induces the transition of EF-Tu from the GTP- to the GDP-bound conformation (k4),35 aa-tRNA accommodation in the A site and peptide bond formation (k5; the accommodation 5859

DOI: 10.1021/jacs.7b00727 J. Am. Chem. Soc. 2017, 139, 5857−5864

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Journal of the American Chemical Society

Fitting of k1 and k−1 relied on data obtained in a model system without a codon in the A site, which yields results equivalent to initial binding to programmed ribosomes, but is technically more robust;33,34,48 additional information is contained in the time courses of cognate AAA decoding. The rate constants of codon recognition were derived from the time courses of fluorescence changes of tRNALys(Prf16/17) upon binding to the cognate AAA codon. Fitting of the k2 and k−2 values was restrained by the measured Kd value of the codon-recognition complex (70 nM and 400 nM for mcm5s2U34 and mcm5U34 Lys-tRNALys, respectively; Figure 1e). The information on the k3 values is contained in the time courses monitored by mantGTP fluorescence changes and of GTP cleavage measured by quench-flow (Figures 1f, 1g, S1, S2). Because ternary complexes were purified from unbound EF-Tu and aa-tRNA, we expected that upon binding to the ribosome all ternary complexes will hydrolyze GTP. The observed GTP hydrolysis was 100% and 60% with mcm5s2U34 and mcm5U34 LystRNALys, respectively. A somewhat reduced end-level of GTP hydrolysis with the hypomodified tRNA is most probably due to the dissociation of a fraction of ternary complexes after purification. The fraction of the active vs total ternary complexes in the mixture was estimated by introducing additional fitting parameters for the active fraction based on the end-levels of GTP hydrolysis. Alternatively, including additional steps that could lead to ternary complex release from the ribosome prior to GTP hydrolysis, such as additional dissociation steps after codon recognition or GTPase activation, did not yield satisfactory fits and was discarded. The step at which Pi is released and the conformational change of EF-Tu takes place (k4) is reported by the fluorescence change of the reporter MDCC-PBP, as well as by the delay between GTP hydrolysis and aa-tRNA accommodation/peptide bond formation and is also reported by the Prf and mant labels. Notably, the end-level of peptide bond formation was lower than that of GTP hydrolysis (Figure 1g,i). This can be attributed to the rejection of aa-tRNA after GTP hydrolysis, but prior to aatRNA accommodation and peptide bond formation (dissociation step k7). The evaluation of the quality of the global fits and the lower and upper boundaries of the rate constants are given in Table S1. The comparison of the rate constants of the elemental steps obtained with wild-type and hypomodified Lys-tRNALys shows how the s2 modification affects decoding. The rate constants of initial binding and codon recognition are similar for mcm5s2U34- and mcm5U34-modified Lys-tRNALys (Figure 2b, Table S1). The stability of the codon-recognition complex is reduced for mcm5 tRNALys compared to the fully modified tRNALys due to a 6-fold higher dissociation rate constant (k−2 = 4 s−1 and 22 s−1) (Figure 2b, Table S1). The calculated rate constants of GTPase activation and GTP hydrolysis (k3) are too high to discern the effect of thiolation. However, we observed that the rate constants of Pi release and EF-Tu dissociation (k4) differ by 7-fold (Figure 2b, Table S1). The rate constants of aa-tRNA accommodation/peptide bond formation before the release of EF-Tu (k5a), and the rate constants of EF-Tu release are largely independent of the thiolation. The rate constant of peptide bond formation after EF-Tu release (k5b) may be slightly (2-fold) reduced in the absence of thiolation, but the effect is not statistically robust (Table S1). In contrast, the rate of Lys-tRNALys dissociation at the proofreading stage (k7) is higher for the tRNA lacking the s2 modification compared to the fully modified tRNA (Figure 2b,

and the chemistry steps are kinetically indistinguishable) with concomitant release of EF-Tu (k6).33,34,36 In view of recent suggestions that the conformational changes of EF-Tu−GDP may accelerate aa-tRNA accommodation,37,38 we introduced separate parallel pathways for aa-tRNA accommodation/ peptide bond formation before (k5a) and after (k5b) EF-Tu release (k6a and k6b, respectively). Aa-tRNA may be rejected from the ribosome (k7) during the proofreading step. Additional aa-tRNA dissociation steps upon codon recognition or GTPase activation were considered and refuted as they did not result in satisfactory data fits. To study the kinetics of AAA codon reading by yeast LystRNA, we used a bacterial translation system. This is justified by the high degree of structural conservation of the ribosome decoding site39−41 and the conservation of the fundamental mechanisms of decoding.42,43 To monitor the kinetics of A-site binding, we used observables that were extensively validated in previous studies.33−35,44 Native tRNALys containing mcm5s2U34 or mcm5U34 modifications was prepared from the wild-type and the urm1Δ yeast strains, respectively,21 and the extent of modification tested as described.11 The ternary complexes EFTu−GTP−Lys-tRNALys were purified by gel filtration and mixed with the ribosome complexes that contained an AAA codon in the A site and fMet-tRNAfMet bound to the AUG codon in the P site. We followed the time courses of reactions in the milliseconds to seconds range by rapid kinetics techniques, stopped-flow for fluorescence reporters and quench-flow for chemical reactions. A fluorescent tRNA derivative, Lys-tRNALys(Prf16/17), containing proflavin at positions 16 or 17 in the D loop was used to monitor tRNALys rearrangement steps (Figures 1b, 1c). To determine the stability of the codon-recognition complex with Lys-tRNALys (mcm5s2U34 or mcm5U34) in the prehydrolysis state, we used the GTPase-deficient mutant EF-Tu(H84A), which behaves as wild-type EF-Tu in initial binding and codon recognition.45 Using the GTPase-deficient EF-Tu mutant, we determined the dissociation rate of the complex (Figure 1d) and the overall affinity (Kd) of the codon recognition complex by fluorescence titrations (Figure 1e). Structural rearrangements of EF-Tu, including the GTPase activation, were monitored using a fluorescent GTP derivative, mant-GTP (Figure 1f).46 GTP hydrolysis and peptide bond formation were measured using [γ-32P]GTP (Figure 1g) and [14C]Lys-tRNALys (Figure 1i), respectively. Pi release (Figure 1h) was monitored using a fluorescence-based reporter system with the phosphate binding protein labeled by a fluorescent reporter 7-diethylamino-3((((2-maleimidyl)ethyl)amino)carbonyl)-coumarin (MDCCPBP).35 For each observable, we carried out experiments at constant concentration of the ternary complex (0.1 μM) and increasing concentrations (0.8−2 μM) of ribosome complexes. The large size of the data set (Figures S1, S2) allowed us to use unbiased global fitting by KinTek Explorer software,47 without any prior knowledge about the fluorescence changes at each step and bypassing the need for exponential fitting and analytical evaluation of the concentration dependencies of apparent rate constants. We used a kinetic model comprising seven steps (Figure 2a), which was the minimum number of steps for the best fit of all data for wild-type tRNALys containing mcm5s2 and hypomodified tRNALys lacking the s2 modification. Initial binding of the ternary complex to the A site results in a conformational rearrangement that can be detected by fluorescence changes of tRNALys(Prf16/17), by analogy with previous data obtained with Phe-tRNAPhe(Prf16/17).34,44,48 5860

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Figure 3. EF-G-induced subunit rotation and translocation. (a) Subunit rotation induced by adding EF-G−GTP to fMetLys-tRNALys PRE complexes with mcm5s2U34 (black) or mcm5U34 (blue) modifications. FRET between Alx488 and Alx568 attached to proteins bS6 and bS9 was monitored by donor fluorescence. Smooth red lines represent two-exponential fits. (b) Apparent rate constants of the CCW (kapp1) and CW (kapp2) of subunit rotation upon translocation calculated from the data in (a). Error bars indicate SEM of three independent experiments. (c) Time-resolved Pmn assay. fMetLys-tRNALys PRE complexes (0.1 μM) were rapidly mixed with EF-G−GTP (2.0 μM) and Pmn (1.25 mM) in a quench-flow apparatus, and the amount of fMetLys-Pmn was determined (Materials and Methods). Smooth red lines represent single-exponential fits. Error bars indicate SEM of three independent experiments. (d) Apparent rate constants of translocation (kapp) calculated from the data in (c). Error bars indicate SEM of three independent experiments. (e) Pmn reaction with POST complexes. Quench-flow experiments were performed as in (c). Smooth red lines represent single-exponential fits.

(CW) rotation takes place concomitantly with the tRNA translocation on the SSU.52 We monitored time courses of EFG-facilitated subunit rotation by stopped-flow, mixing pretranslocation complexes (PRE) containing fMetLys-tRNALys in the A site and deacylated tRNAfMet in the P site with EF-G− GTP at saturating concentration. Upon addition of EF-G to the PRE complexes, FRET changed in a biphasic fashion, with distinct downward and upward phases reporting on the CCW and CW rotation,51 respectively (Figure 3a). The rate constant of the CCW rotation was not altered by the lack of thiolation (Figure 3b, Table S2). In contrast, the rate of the CW rotation was significantly slower with tRNALys lacking s2 (Figure 3b, Table S2). Second, we monitored translocation on the LSU by the timeresolved puromycin (Pmn) assay (Figures 3c, 3d, 3e). Pmn can react with fMetLys-tRNALys only after it has been translocated to the P site; because the Pmn reaction intrinsically is rapid,51,53 kPmn provides a good estimate for the tRNA translocation rate. For EF-G−GTP induced translocation, kPmn was 2 s−1 and 1 s−1 for mcm5s2U34 and mcm5U34 tRNALys, respectively (Figure 3d, Table S2). As a control, we performed the Pmn reaction with post-translocation (POST) complexes containing fMetLystRNALys in the P site. These data show that the intrinsic rate

Table S1). Thus, thiolation at U34 affects the stability of LystRNALys binding to the ribosome at both tRNA selection steps (k−2 and k7) and modulates the rate constant (k4) of the step that is associated with Pi release and the rearrangement of EFTu from the GTP- to the GDP-bound conformation. We note that the time courses of dipeptide formation reported here are almost identical to those used in our earlier work to estimate the kpep values.21 In contrast to the elemental rate constants presented here, the kpep value represents an overall effect on peptide bond formation. The lower stability of tRNA binding in the absence of s2 observed here is also consistent with the previous study.21 Impeded Translocation with Hypomodified tRNALys. To monitor EF-G-induced tRNA−mRNA translocation, we used two sets of observables. First, we monitored the rotation of the small ribosomal subunit (SSU) relative to the large ribosomal subunit (LSU) monitoring FRET (fluorescence resonance energy transfer) between a pair of fluorophores attached to ribosomal proteins bS6 and bL9 (Figures 3a, 3b).49−51 After peptide bond formation, SSU spontaneously rotates in the counterclockwise (CCW) direction relative to the LSU. EF-G accelerates the CCW rotation and shifts the equilibrium toward the rotated state.51 The reverse, clockwise 5861

DOI: 10.1021/jacs.7b00727 J. Am. Chem. Soc. 2017, 139, 5857−5864

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Journal of the American Chemical Society of fMetLys-Pmn formation was unaffected by the tRNA modification (Figure 3e). These data together indicate that the loss of thiolation impedes tRNA translocation.

modification may affect this process by altering the conformational dynamics of tRNA on the ribosome, e.g., by changing the energetic preferences of spontaneous fluctuations between the distorted state and relaxed state. The elemental rates of decoding and translocation (Tables S1 and S2) allow us to predict how long the ribosome remains on a Lys codon during elongation (Figure S3). The increased dissociation rate of s2-deficient Lys-tRNALys at the initial selection stage (k−2) reduces the affinity of the ternary complex to its cognate codon, but because the rate constant of the GTPase activation (k3) is much higher than the dissociation rate constant (k−2), the observed rate of GTP hydrolysis at the first round of initial selection is not changed appreciably (Figure S3a). However, when tRNA is rejected during the proofreading step (k7), the decoding cycle has to be repeated. This results in an excessive GTP hydrolysis, which is higher for the hypomodified, than for the fully modified, tRNALys. The posthydrolysis rearrangement step (k4) is much slower with the hypomodified tRNALys. The delay in the Pi release and the tRNA drop-off result in slower decoding as seen from the peptide bond formation. We estimate that the lack of s2 modification increases the time required to decode 50% of Lys codons from 1 to 3 s (Figure S3a). The slower translocation of mcm5U34 tRNALys compared to the fully modified tRNA further contributes to ribosome pausing. The total time for the elongation cycle increases from 2.5 s to about 3.5 s (Figure S3b); thus, the residence time of the ribosomes on Lys codons is expected to increase by 40%. This nicely accounts for the modest (20−40%) increase in the ribosome occupancy of the AAA codons in the ribosome profiling experiments.22,23 Overexpression of unmodified tRNAs in the urm1Δ strain may compensate for its lower affinity and ensure a more rapid rerecruitment of the ternary complex when the tRNA is rejected during proofreading; this explains the rescue effect of overexpression of hypomodified tRNA in the urm1Δ strain.21 Thus, the measured effects on the elemental rates of translation elongation account for the observed accumulation of ribosomes on the AAA codons in urm1Δ strains in vivo. In summary, the present kinetic data explain how moderate changes in elemental reactions of decoding and translocation can lead to substantial changes in translation elongation at specific codons and to increased frameshifting. Thus, modifications at U34 tRNA appear to play an important role in maintaining the exact rhythm of translation.

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DISCUSSION Our kinetic analysis provides a comprehensive description of the recognition, decoding and translocation mechanisms for mcm5s2U34 and mcm5U34 tRNALys. The elemental rate constants of EF-Tu-dependent AAA decoding by the fully modified mcm5s2U34 Lys-tRNALys are remarkably similar to those of other tRNAs for which the kinetic mechanisms was established, e.g., tRNAPhe and tRNA1BAla,34,54 supporting the notion that tRNA sequences and modifications evolved in a way to ensure a uniform rate of decoding.55 The main difference between tRNAs are different values of k−2 that characterizes the stability of the codon-recognition complex. The dissociation rate constant varies between 0.2 s−1 for tRNA Phe (codon-anticodon base pairing UUU:AAG or UUC:AAG), 1 s−1 for tRNA1BAla (GCC:CGU** where U** is uridine 5-oxyacetic acid (cmo5U34) modification)34,54 and 4 s−1 for fully modified tRNALys (AAA:UUU*, U* = mcm5s2U) (this paper). This comparison demonstrates that the stability of the codon-recognition complex is independent of the G-C content of the codon-anticodon complex. This supports the notion that the ribosome senses the correct geometry of the codon-anticodon complex, whereas the number of hydrogen bonds is less important.5,56,57 The lack of the s2 modification further increases the dissociation rate of the codon-anticodon complex, which is manifested both at the initial-selection and proofreading phases (k−2 and k7). In the crystal structure of ribosome complex with E. coli tRNA Lys in the fully accommodated state the s2 group is involved in the stabilization of the conformation of the anticodon loop through stacking with U35, but does not seem to interact with neighboring groups of the ribosome. In the absence of the s2 group, the anticodon loop may adopt a somewhat different conformation; the higher dissociation rate may reflect the energetic cost for inducing the conformation favorable for decoding. The reduced stability of the codon-anticodon interaction, in combination with slow translocation, can also stimulate frameshifting.58 In fact, yeast strains defective in tRNALys thiolation show increased propensity for +1 frameshifting in the A site.24 Thus, it is likely that reduced stability of the codon-anticodon complex and enhanced tRNA drop-off from the cognate codon increase the probability of frameshifting. One surprising observation is a 7-fold reduction in the k4 value with Lys-tRNA lacking the s2 modification. Previous experiments with tRNAPhe suggested that k4 entails posthydrolysis rearrangements of EF-Tu, including Pi release and the conformational change from the GTP to the GDP-bound form.34 It is difficult to envisage how the lack of a modification at the wobble position can modulate these reactions, as the trigger of Pi release and EF-Tu rearrangement is not known. In the GTPase-activated state the CCA end of the tRNA is still tightly bound to the factor with aa-tRNA body adopting in a distorted conformation, as deduced from the structures of EFTu−GDPCP59,60 or a EF-Tu-like GTPase SelB−GNPNP31 bound to the ribosome. In solution, distorted and nondistorted tRNA conformations are likely isoenergetic.31 The propensity of the tRNA to adopt the relaxed conformation may drive the release of the tRNA CCA end from EF-Tu, thereby initiating the conformational switch allowing for the Pi release and the transition of EF-Tu to the GDP form. The lack of the s2

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MATERIALS AND METHODS

All experiments were carried out at 24 °C in buffer A (TAKM7) (50 mM Tris-HCl pH 7.5, 50 mM NH4Cl, 50 mM KCl, 7 mM MgCl2, 1 mM DTT). Ribosomes, EF-Tu, and fMet-tRNAfMet from E. coli were prepared as described.34,46,61 Labeling of ribosomal proteins bS6 and bL9 with Alexa488 and Alexa568 was carried out as described.51 The mRNA (28 nt long) with a AAA codon following the AUG start codon was purchased from IBA (Germany). The preparation of total tRNA and the purification of [14C]Lys-tRNALys from wild-type S288C and urm1Δ yeast strains was carried out as described.21 Prf-labeled yeast tRNALys was prepared as described previously for tRNAPhe.62 The extent of the s2 modification was verified by ((N-acryloylamino)phenyl)mercuric chloride (APM)-gel retardation analysis of tRNA11 and was 70−90% for native tRNALys and 2% for tRNALys from the urm1Δ strain, respectively. The presence of mcm5 modification in the absence of s2 was shown in previous reports.63 Ternary complex EF-Tu−GTP−[14C]Lys-tRNALys(Prf16/17) was prepared by incubating EF-Tu (50 μM), GTP (1 mM), phosphoenol pyruvate (3 mM), pyruvate kinase (0.05 mg/mL), tRNALys (Prf16/17) (25 μM), CTP (1 mM), ATP (3 mM), L-[14C]lysine (40 μM), and 5862

DOI: 10.1021/jacs.7b00727 J. Am. Chem. Soc. 2017, 139, 5857−5864

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Journal of the American Chemical Society Lys-tRNA synthetase (2% v/v) in buffer A and purified by gel filtration on two Superdex 75 HR columns operated in tandem (GE Healthcare). The ternary complex of EF-Tu(H84A) was prepared according to the same procedure. Initiation and pretranslocation (PRE) complexes were prepared as described.52,64−66 Initiation complexes were formed in buffer A by incubating ribosomes (1 μM), mRNA (3 μM), f[3H]Met-tRNAfMet (1.5 μM), IF1, IF2, IF3 (2 μM each), and GTP (1 mM) for 30 min at 37 °C, and purified by centrifugation through 400 μL 1.1 M sucrose cushion in buffer A for 2 h at 4 °C and 259 000g in a Beckman Optima Max-XP ultracentrifuge. After centrifugation, pellets were dissolved in buffer A, shock-frozen in liquid nitrogen and stored at −80 °C. When fluorescence labeled ribosomal subunits were used, the SSU with bS6 labeled by Alexa488 was first heat-activated in TAKM with 21 mM MgCl2 for 30 min at 37 °C, combined with a 1.5-fold excess of labeled LSU (with bL9 labeled with Alexa568) and incubated with a 3-fold excess of mRNA and a 2-fold excess of IF1, IF2, IF3 each and a 2.5fold excess of f[3H]Met-tRNAfMet in TAKM7 buffer containing 1 mM GTP for 30 min at 37 °C. To prepare PRE complexes, the initiation complexes were mixed with a 2-fold excess of ternary complex and incubated for 1 min at 37 °C and the complexes were purified by centrifugation through 1.1 M sucrose cushion in TAKM containing 21 mM MgCl2. Pellets were resuspended in the same buffer and the amount of bound aa-tRNA was determined by nitrocellulose filtration. Rapid Kinetics. Fluorescence stopped-flow experiments were performed in a SX-20MV spectrometer (Applied Photophysics). Fluorescence of Prf was excited at 463 nm and of mant at 363 nm, and measured after passing KV500 or KV408 long-pass filters (Schott), respectively. Experiments were performed by rapidly mixing equal volumes of reactants and monitoring the time courses of fluorescence changes. Time courses depicted in the figures were obtained by averaging 5−7 individual traces. When working with fluorescencelabeled ribosomes (S6Alx488−L9Alx568), Alx488 was excited at 470 nm and the fluorescence of Alx568 was monitored after passing through a KV590 cutoff filter (Schott). EF-G-induced SU rotation was monitored after rapidly mixing PRE complexes (0.1 μM) with EF-G (4 μM) in buffer A. Pi release from EF-Tu after GTP hydrolysis was monitored by the fluorescence change of MDCC-PBP.35 Initiation complexes and purified ternary complexes were rapidly mixed in a stopped-flow apparatus. Both reaction mixtures contained MDCC-PBP (2.5 μM), purine nucleoside phosphorylase (0.1 U/mL), and 7-methylguanosine (200 μM) (the latter two components serving as “Pi mop” to take up trace amounts of contaminating Pi).67 MDCC fluorescence was excited at 425 nm and measured after passing a cutoff filter (KV 450, Schott). The rates of GTP hydrolysis and peptide bond formation were measured by quench-flow in a KinTek RQF-3 apparatus. To measure GTP hydrolysis, reactions were quenched with 50% formic acid and the fraction of [γ-32P]GTP hydrolyzed was analyzed by thin layer chromatography.34 To determine the rates of peptide bond formation, reactions were quenched with KOH (0.5 M); peptides were released by alkaline hydrolysis for 45 min at 37 °C, separated by reversed-phase HPLC (LiChrospher 100 RP-8, Merck), and quantified by doublelabel radioactivity counting.53 To additionally validate the end level of reactions, GTP hydrolysis and peptide bond formation was measured after 10, 20, and 30 s in multiple replicates by hand pipetting. Fluorescence Titrations and Data Evaluation. Fluorescence titrations were performed on a Fluorolog-3 fluorimeter (Horiba). Ternary complex EF-Tu(H84A)−GTP−[14C]Lys-tRNALys(Prf) (40 nM) was mixed with programmed ribosomes (0−350 nM) and the change in Prf fluorescence was monitored. Prf was excited at 456 nm and the emission was measured at 505 nm. The data were fitted with a quadratic equation to calculate Kd values.68 Time-Resolved Pmn Assay. The rate of translocation was measured by the time-resolved Pmn assay.65 PRE complex (0.1 μM) was rapidly mixed with Pmn (2.5 mM) and EF-G (2 μM) in buffer A at 24 °C. Control experiments were carried out with POST complexes prepared by incubating PRE complexes with EF-G. POST complexes with fMetLys-tRNALys (0.1 μM) in the P site were rapidly mixed with

Pmn (1.25 mM) in a quench-flow apparatus. The reaction was quenched with KOH (0.5 M) and the peptides were released by alkaline hydrolysis for 45 min at 37 °C, analyzed by reversed-phase HPLC (LiChrospher 100 RP-8, Merck), and quantified by doublelabel f[3H]Met-[14C]Lys radioactivity counting.53 Data Analysis. Exponential fitting was performed with GraphPad Prism, numerical integration with KinTek Explorer.69 The goodness of the global fits was evaluated using the statistical tools developed to evaluate multidimensional parameter space in fitting kinetic data70 and implemented in KinTek Explorer.47

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b00727. Global fitting of time courses, the goodness of global fitting for mcm5s2U34 and mcm5U34 tRNALys, and a simulation of the ribosome residence time on Lys codons (PDF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Marina V. Rodnina: 0000-0003-0105-3879 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Heena Sharma for providing fluorescence-labeled ribosomes, Wolfgang Wintermeyer for critical reading, and Theresia Niese, Olaf Geintzer, Sandra Kappler, Christina Kothe, Anna Bursy, Tanja Wiles, and Michael Zimmermann for expert technical assistance. This work was supported by Deutsche Forschungsgemeinschaft (DFG) in the framework of the Sonderforschungsbereich SFB860.

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