- Email: [email protected]
Adaptation of aminoacylation identity rules to mammalian mitochondria
Biochimie 94 (2012) 1090e1097
Contents lists available at SciVerse ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
Adaptation of aminoacylation identity rules to mammalian mitochondria Aurélie Fender, Agnès Gaudry, Frank Jühling, Marie Sissler, Catherine Florentz* Architecture et Réactivité de l’ARN, CNRS, Université de Strasbourg, IBMC, 15 rue René Descartes, F-67084 Strasbourg Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 January 2012 Accepted 23 February 2012 Available online 2 March 2012
Many mammalian mitochondrial aminoacyl-tRNA synthetases are of bacterial-type and share structural domains with homologous bacterial enzymes of the same specificity. Despite this high similarity, synthetases from bacteria are known for their inability to aminoacylate mitochondrial tRNAs, while mitochondrial enzymes do aminoacylate bacterial tRNAs. Here, the reasons for non-aminoacylation by a bacterial enzyme of a mitochondrial tRNA have been explored. A mutagenic analysis performed on in vitro transcribed human mitochondrial tRNAAsp variants tested for their ability to become aspartylated by Escherichia coli aspartyl-tRNA synthetase, reveals that full conversion cannot be achieved on the basis of the currently established tRNA/synthetase recognition rules. Integration of the full set of aspartylation identity elements and stabilization of the structural tRNA scaffold by restoration of D- and T-loop interactions, enable only a partial gain in aspartylation efficiency. The sequence context and high structural instability of the mitochondrial tRNA are additional features hindering optimal adaptation of the tRNA to the bacterial enzyme. Our data support the hypothesis that non-aminoacylation of mitochondrial tRNAs by bacterial synthetases is linked to the large sequence and structural relaxation of the organelle encoded tRNAs, itself a consequence of the high rate of mitochondrial genome divergence. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Identity elements Mitochondrial tRNA Cross-aminoacylation Structural plasticity
1. Introduction Rules governing interaction of tRNAs and aminoacyl-tRNA synthetases (aaRSs1) for efficient and specific aminoacylation have been well established for a large number of aminoacylation systems in the three kingdoms of life (for reviews, see [1e4]). They rely on identity nucleotides, mainly located at both extremities of the tRNA L-shaped 3D structure that contact specific amino acids in the aaRS structure. Identity sets are composed of major and minor elements, with major elements usually conserved during evolution, as is the architecture of tRNA. Accordingly, it became possible to convert the specificity of a given tRNA by simple transplantation of identity sets, and/or by removal of antideterminants (signals hindering recognition by a synthetase of other specificity). Cryptic signals and permissive nucleotides that also contribute to specificity were discovered in this context [5,6]. Cross-species aminoacylation of tRNAs from a given organism by synthetases of the same specificity but from other organisms were reported in great numbers [1]. Exceptions where cross-aminoacylation was not
* Corresponding author. Tel.: þ33 (0) 3 88 41 70 59; fax: þ33 (0) 3 88 60 22 18. E-mail address: [email protected] (C. Florentz). 1 aaRS stands for aminoacyl-tRNA synthetase with aa for amino acid (for individual aaRS, aa is given in the 3 letter code, e.g. AspRS for aspartyl-tRNA synthetase). 0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2012.02.030
possible were explained by the presence of only partial sets of identity elements in the tRNA or by the existence of alternate sets, and in many cases could be overcome by simple mutagenic approaches in vitro [1]. An unsolved cross-aminoacylation barrier concerns mammalian mitochondrial (mt) tRNAs which are not aminoacylated by bacterial synthetases [7,8]. In mammalian mitochondria, aminoacylation systems are of dual origin with the tRNAs coded by a simplified and rapidly evolving mt-genome and aaRSs coded by the slowly evolving nuclear genome and imported into mitochondria. Mammalian mtaaRSs have various evolutionary origins [9] but many are of bacterial-type ([10,11] and references therein) and do not depart from the already known synthetases. The human mt-aaRSs (Hsa mtaaRS) have no drastic apparent structural peculiarities as compared to their evolutionarily-related bacterial counterparts (except PheRS, which is a monomer instead of an a2b2 tetramer), i.e. they have the same structural organization, possess the expected class-specific signature motifs in their catalytic domains and, accordingly, belong to the expected aaRS-class [12,13]. However, Kumazawa and colleagues [7,8] reported already twenty years ago on aminoacylation of Escherichia coli (Eco) and Thermus thermophilus (Tth) tRNAs by bovine mt-aaRSs specific for threonine, arginine, lysine, phenylalanine and serine, but absence of charging of bovine (Bos taurus or Bta) mt-tRNAs by the corresponding bacterial aaRSs (Table 1). This is intriguing since three of these enzymes (Bta mt-
A. Fender et al. / Biochimie 94 (2012) 1090e1097
2.2. tRNA gene mutagenesis and in vitro transcription
Table 1 Unilateral aminoacylation of mammalian mitochondrial tRNAs. aa
tRNA
aaRS Bta mt
Eco
Tth
Phe
Bta mt Eco Tth Bta mt Eco Tth Bta mt Eco Tth Bta mt Eco Tth Bta mt Eco Tth
YES YES YES YES YES YES YES YES WEAK YES Hsa mt YES YES
NO þ NO þ WEAK þ NO þ NO þ Eco NO nt
NO þ NO þ NO þ NO NO þ Tth NO þ
Thr
Arg
Lys
Ser
Asp
Hsa mt Eco Tth
1091
Ref
[7]
[8]
[8]
[8]
[8]
[14]
Only aminoacylation capacities for couples involving a single mitochondrial partner, either mitochondrial tRNA or mitochondrial aminoacyl-tRNA synthetase, are emphasized by YES (active), NO (inactive) or WEAK (partially active). Aminoacylation capacities of other couples are indicated as follows: positive control activities of cognate couples (); aminoacylation activity for systems involving both bacterial tRNA and bacterial synthetase (þ/); non tested (nt). Bta mt: Bos taurus mitochondrial; Hsa mt: Homo sapiens mitochondrial; Eco: Escherichia coli; Tth: Thermus thermophilus.
ThrRS, -PheRS and -SerRS) are of the bacterial-type [9,11]. A similar barrier was observed for the Hsa mt-aspartylation system from which the tRNA is not recognized by a bacterial synthetase, i.e. either Eco AspRS or Tth AspRS, while the Hsa mt-AspRS, which is of prokaryotic-type, recognizes and aminoacylates both Eco and Tth tRNAAsp ( [14]; our unpublished results) (Table 1). These data demonstrate unilateral aminoacylation of mt-tRNAs by mt-aaRSs, and show a strong cross-species barrier for aminoacylation of mttRNAs by bacterial synthetases. To sort out the possible molecular reasons for this barrier, the specific case of non-aminoacylation of Hsa mt-tRNAAsp by the Eco AspRS was investigated as a model system by a mutagenic in vitro approach. As a result, we have observed that the absence of classical aminoacylation signals, namely identity and structural elements, are key elements for non-cross-aminoacylation of the mt-tRNA, but that the intrinsic unusually large thermodynamically-driven structural plasticity of the mt-tRNA also forms an important element in the non-recognition barrier. We propose that nonaminoacylation of mammalian mt-tRNAs by bacterial synthetases is linked to the global divergence of the organelle encoded tRNAs, far beyond loss of identity elements and well-defined structural features, a consequence of the very high mutation rate of mitochondrial genomes. 2. Material and methods
Hsa mt-tRNAAsp variants displaying individual Eco identity elements [18,19] (mt-tRNAAspG73, mt-tRNAAspC38 and mttRNAAspG2eC71) or “classical” D- and T-loops as found in tRNAAsp from Schizosaccharomyces pombe (mt-tRNAAspDT) have been prepared previously [14]. Hsa mt-tRNAAsp variant (G73/DT, C38/G73, idec,2 idec/DT, idec/U11eA24, and idec/DT/U11eA24) genes were obtained by hybridization of 9 overlapping oligonucleotides, ligation between BamHI and HindIII sites of plasmid pTFMa [20]. The gene for variant idec/DT/U11eA24/C31eG39 was derived from the idec/DT/ U11eA24 gene with the QuikChangeÔ Site-Directed Mutagenesis Kit (Stratagene). All these genes contain a hammerhead ribozyme [21] and tRNA sequences downstream from the T7 polymerase promoter. A BstNI site coincidental with the 30 -end of the tRNA sequences allows synthesis of tRNAs ending with the expected CCA sequence. Eco tRNAAsp variant (mtDT and A11eU24) genes were obtained by PCR amplification using two complementary 30 nt-long primers (allowing for introduction of BamHI and HindIII sites) that contain the T7 polymerase promoter and tRNA sequences. All synthetic genes were transformed into TOP10 cells (Invitrogen). Transcription was as described [21]. tRNAs were purified to single nucleotide resolution on denaturing polyacrylamide gels as described [14]. Quality of tRNAs has been verified on native PAGE which revealed only single populations of conformers. 2.3. E. coli and human mt-AspRS Eco AspRS was a gift from G. Eriani (IBMC, Strasbourg). Hsa mtAspRS was expressed and purified on a nickel affinity column [10]. Protein concentrations were determined from OD280 using the extinction coefficients (ε(Eco$AspRS) ¼ 42985 M1 cm1 and ε(Hsa$mt1 cm1) and molecular weights (MW(Eco$AspRS) AspRS) ¼ 43540 M ¼ 65913.4 g mol1 and MW(Hsa$mt-AspRS) ¼ 69627 g mol1) calculated with ProtParam from ExPASy tools (expasy.org). 2.4. Aminoacylation of tRNAs Aminoacylation assays were performed as described [14]. Assays were conducted at 15 C in 50 mM HEPES-KOH pH 7.5, 25 mM KCl, 12 mM MgCl2, 2.5 mM ATP, 0.2 mg/mL BSA, 1 mM spermine, 32 mM [3H]-aspartic acid (208 GBq/mmol), and adequate amounts of transcripts and aaRS. Assays were performed in 50 mL samples. Transcripts were renatured at 60 C (for mt-tRNAAsp and variants) or 85 C (for Eco tRNAAsp and variants) for 90 s in water and slow cooling down to room temperature before aminoacylation. Maximal charging levels (plateaus) were determined for 400 nM transcript and optimal concentrations of synthetase (500 nM Hsa mt-AspRS or 200 nM Eco AspRS). Kinetic parameters kcat and KM were derived from LineweavereBurk plots obtained using a range of tRNA concentration from 200 nM to 8 mM (the very high KM of 70 mM for one variant is an approximation) and of Eco AspRS from 2 to 100 nM. Experiments were performed at least in triplicate on separate preparations of transcripts. Final kcat and KM values varied up to 40%.
2.1. Materials Synthetic genes of wild-type Eco tRNAAsp [15] and Hsa mttRNAAsp [10] were cloned previously. T7 RNA polymerase was purified as described [16]. Tth AspRS (discriminating version [17]) and the corresponding in vitro transcribed tRNAAsp were gifts from H.D. Becker and D. Kern (IBMC, Strasbourg). Oligonucleotides were from Sigma Genosys, restriction enzymes (BamHI, HindIII, and BstNI) from New England Biolabs, T4 DNA ligase from Qbiogen, and 3 L-[ H] aspartic acid (208 GBq/mmol) from Amersham.
2.5. Thermodynamic stability of tRNAs Free energies (expressed as kcal/mol) of tRNA structures were calculated with the RNAeval program of the Vienna RNA package (http://rna.tbi.univie.ac.at/cgi-bin/RNAeval.cgi), with fixed secondary
2 Idec stands for identity elements of Eco tRNAAsp for aspartylation by Eco AspRS, namely G73, G2eC71, G10, G34, U35, C36, C38.
1092
A. Fender et al. / Biochimie 94 (2012) 1090e1097
structure constraints and with a temperature of 15 C. Advanced options “dangling energies on both sides of a helix in any case” and “RNA parameters (Turner Model, 2004)” were applied. 3. Results and discussion 3.1. Characteristic features of heterologous Hsa mt-tRNAAsp and Eco AspRS Hsa mt-AspRS is of the prokaryotic-type, with the typical bacterial insertion domain and C-terminal extension, and shares 43% of strictly identical residues with Eco AspRS [10]. The human mt-AspRS possesses residues strictly conserved in all known AspRS sequences which include residues involved in ATP-, tRNA-, and amino acid-binding (those typical for class II aaRSs and those specific for aspartic acid recognition) [10]. While the two enzymes are similar, the corresponding tRNAs are markedly different. Hsa mt-tRNAAsp (Fig. 1) is a typical example of mammalian mt-tRNA in terms of sequence and structural features [22] and is to a large extent distinct from Eco tRNAAsp (Fig. 1). It has a highly skewed A, U and C nucleotide content (90% versus 66%), a high content of weak AeU and GeU base-pairs (16 out of 21 versus 7 out of 21) and nonclassical D- and T-loops. In terms of 3D structures, both tRNAs are however of the same type, with a common network of core interactions but with a major difference in the elbow of the L-shape where D/T-loop interactions stabilize the Eco tRNAAsp but are absent in the mt-tRNAAsp [23]. The two tRNAs are typical structural
representatives of bacterial tRNAs and mammalian mt-tRNAs, respectively [24]. In terms of signals responsible for aspartate aminoacylation specificity, Eco tRNAAsp possesses the major aspartate determinants identified in all “classical” (i.e. non organelle) aspartate systems investigated so far (reviewed in [1,4]), namely the discriminator residue G73 (a primordial element, sufficient for specific aminoacylation of a minihelix mimicking the acceptor branch of an L-shaped tRNA, [25]), the anticodon triplet G34U35C36 and base-pair G10eC25 (U25 is also tolerated). Eco AspRS is also sensitive to additional minor elements, namely residue C38 and base-pair G2eC71 [19,26]. By contrast, in the Hsa mt-tRNAAsp, the aspartate identity set is restricted to the anticodon triplet G34U35C36 where only U35 and G36 are major determinants [14]. This minimal set is sufficient for specific aspartylation by Hsa mt-AspRS. Notably, the discriminator residue 73 and minor elements such as base-pair G10eC25 are not involved. The existence of a restricted set of aspartate identity elements is valid all over mammalian mt-tRNAAsp [27]. As a consequence of these properties, a trivial hypothesis for the non-aspartylation of the mt-tRNA by Eco AspRS would be the absence of identity elements G73, G2eC71 and C38, and the absence of a structural determinant that would provide a stable D/T-loop interaction, as suggested previously for the cases presented in Table 1 [8]. In what follows, the contribution of these elements has been investigated by a mutagenic approach aimed at progressively converting mt-tRNAAsp into an efficient substrate for aspartylation by Eco AspRS.
Fig. 1. Attempts to convert Hsa mt-tRNAAsp into an efficient substrate for Eco AspRS through application of classical aminoacylation identity rules. A. Variants of Hsa mt-tRNAAsp highlighting progressive incorporation of bacterial tRNAAsp signature motifs. Newly inserted mutations into the global background of human mt-tRNAAsp (grey line) are explicitly indicated; “idec” refers to the identity elements of Eco tRNAAsp. The aminoacylation efficiency by Eco AspRS is indicated (L for loss in aminoacylation efficiency as compared to Eco tRNAAsp for which L ¼ 1) as well as the incidence of the mutations on individual kinetic parameters kcat and KM. G stands for gain in aminoacylation efficiency between two variants. All values are calculated from data presented in Table 2 according to definitions in the legend of Table 2. B. Reference tRNAs, Hsa mt-tRNAAsp wild-type transcript (B1); Eco tRNAAsp wild-type transcript (B2). Eco aspartate identity elements are circled (dark circle for major elements, light grey for minor elements). Tertiary interactions leading to the final Lshaped 3D structures are indicated by dotted lines [24].
A. Fender et al. / Biochimie 94 (2012) 1090e1097
3.2. The full aspartate identity set is necessary but not sufficient for efficient cross-aminoacylation of mt-tRNAAsp In an attempt to convert the human mt-tRNAAsp into a more efficient substrate for Eco AspRS, transcripts with the missing Eco identity elements G73, C38 and base-pair G2eC71 transplanted into them, either individually or in combination, were prepared and tested with both Hsa mt-AspRS and Eco AspRS (Fig. 1). Charging levels are presented in Supplementary Fig. S1, and kinetic parameters in Table 2. While all variants remain fully active for aspartylation by Hsa mt-AspRS as tested previously [14], only the variant hosting the full set of Eco aspartate identity elements, mt-tRNAAspidec (idec for identity element from Eco tRNAAsp) (Fig. 1), became detectably charged by the bacterial enzyme. The other mt-tRNAAsp derivatives, and especially the one including residue G73, remained inactive with charging levels lower than 1%. Establishment of kinetic parameters (Table 2) revealed that kcat is w70-fold lower and KM w100-fold increased as compared to the aminoacylation features of wild-type Eco tRNAAsp transcript. As a consequence, mttRNAAspidec is 7400-fold less efficiently aminoacylated than the wild-type Eco tRNAAsp. According to these data, transplantation of the full set of Eco identity elements is necessary but decisively insufficient for conversion of Hsa mt-tRNAAsp into an efficient substrate for Eco AspRS. 3.3. The full network of tertiary interactions is an additional important but still insufficient feature Most mammalian mt-tRNAs do not display any obvious interaction between their D- and T-loops, allowing great flexibility in the angle between the two branches that form the L-shaped 3D structure [24]. This flexibility could lead to an unusual range of possible distances between the distal extremities of the tRNA, namely the anticodon- and the CCA-end. To evaluate if Eco AspRS is sensitive to this structural feature, classical D- and T-loops (from S. pombe tRNAAsp, which were investigated previously [14]), were transplanted into the initial wild-type mt-tRNAAsp, replacing the smaller and partially degenerated mt-loops, as well as into mttRNAAspidec, with the expectation that they would enable canonical
1093
tertiary interactions and formation of a stabilized elbow of the Lshaped tRNA. According to crystallographic structures, D/T-loop interactions stabilize the angle formed by the acceptor arm and the anticodon arm of the tRNA 3D structure [24]. While the resulting variants mt-tRNAAspDT and mt-tRNAAspidec/DT were as good substrates as the wt mt-tRNAAsp for mt-AspRS [14], mttRNAAspidec/DT (Fig. 1) undergoes a significant improvement as a substrate for Eco AspRS (Supplementary Fig. S1; Table 2). A 4-fold gain in efficiency is observed when compared to mt-tRNAAspidec activity, mainly due to a 4-fold decrease in KM. This modest but significant improvement shows that Eco AspRS not only requires a full aspartate identity set but also is sensitive to the nature of the D- and T-loops of its tRNA substrate. However, the aspartylation capacity of mt-tRNAAspidec/DT remains still 1850-fold less efficient than that of the homologous bacterial tRNAAsp. The importance of the D/T-loop interaction for aspartylation by the Eco enzyme holds true also in the Eco tRNAAsp framework; replacement of Eco tRNAAsp D- and T-loops by the corresponding mt-domains (variant Eco tRNAAspmtDT, Supplementary Fig. S2) leads to a 10-fold decrease in kcat and a 6.5-fold increase in KM for aspartylation with the Eco synthetase. This variant is 70-fold less efficiently aminoacylated by Eco AspRS than wild-type Eco tRNAAsp (Table 2). 3.4. Role of idiosynchratic base-pair A11-U24 in human mt-tRNAAsp Since simple transplantation of identity elements and correction of a structural defect in Hsa mt-tRNAAsp do not allow for full recognition by Eco AspRS, the role of the context elements and especially the presence of antideterminant(s) or neutral elements were considered. A consensus sequence of all tRNAs proven experimentally to be efficient substrates of Eco AspRS in vitro was established and compared to Hsa mt-tRNAAspidec/DT (Supplementary Fig. S3). Base-pair 11e24 has been pinpointed as possible candidate. Hsa mt-tRNAAsp bears A11eU24 while all classical tRNAs have a semi-conserved Y11eR24 pair (Y for pyrimidine, R for purine) [28]. Further, U11 of Eco tRNAAsp makes two hydrogen bonds (one through the base and one through the ribose) with His114 and Asn116 respectively of Eco AspRS (hinge region), as shown in the crystallographic structure of the tRNAAsp/AspRS
Table 2 Kinetic parameters for tRNA aminoacylation by Eco AspRS. tRNA Asp
Wild-type Eco tRNA
Human mitochondrial tRNAAsp variants Wild-type Hsa mt-tRNAAsp Hsa mt-tRNAAspG73 Hsa mt-tRNAAspC38 Hsa mt-tRNAAspG2eC71 Hsa mt-tRNAAspC38/G73 Hsa mt-tRNAAspidec Hsa mt-tRNAAspDT Hsa mt-tRNAAspidec/DT Hsa mt-tRNAAspidec/U11eA24 Hsa mt-tRNAAspidec/DT/U11eA24 Hsa mt-tRNAAspidec/U11-A24/DT/C31eG39 E. coli tRNAAsp variants Eco tRNAAspA73a Eco tRNAAspA38b Eco tRNAAspC2eG72b Eco tRNAAspmtDT Eco tRNAAspA11eU24
kcat (103 s1)
KM (mM)
kcat/KM (103 s1 mM1)
L
2590 320
0.7 0.1
3685
1
nd nd nd nd nd 39 5 nd 34 3 43 14 23 5 319 75
nd nd nd nd nd 69.3 9 nd 17.5 2.5 7.5 3.5 1.6 0.3 55 9
nd nd nd nd nd 0.5 nd 2.0 5.7 14.4 5.8
nd nd nd nd nd 7400 nd 1850 650 260 635
e e e 248 90 3732 340
e e e 4.6 1.8 4.0 2.0
e e e 53.9 933
555 6 5 70 4
In vitro aminoacylation assays were performed as described in “Material and methods” at 15 C. This temperature was found as an optimal compromise between mt-tRNA stability and enzymatic activity of Eco AspRS [14]. nd, not detectable. L stands for relative loss in aminoacylation efficiency considering wt Eco tRNAAsp as the reference (L ¼ (kcat/KM(Eco$tRNA Asp))/(kcat/KM(other tRNA))). a Data from [18]. b Data from [19].
1094
A. Fender et al. / Biochimie 94 (2012) 1090e1097
complex [29]. Therefore, A11eU24 has been replaced by U11eA24 in tRNAAspidec and tRNAAspidec/DT, leading to tRNAAspidec/ U11eA24 and tRNAAspidec/DT/U11eA24 (Fig. 1). Interestingly, both molecules become significantly better substrates for Eco AspRS and remain fully active for Hsa mt-AspRS (Supplementary Fig. S1). tRNAAspidec/U11eA24 and tRNAAspidec/DT/U11eA24 have catalytic efficiencies only 650-fold and 260-fold lower than the wt Eco tRNAAsp, respectively (Table 2). When compared to parental host tRNAs (tRNAAspidec and tRNAAspidec/DT), introduction of the semi-conserved Y11eR24 pair led to improvements in aminoacylation efficiency of 11- and 7-fold, respectively, mainly due to an 11-fold decrease in KM. Thus, in the framework of mttRNAAsp, base-pair A11eU24 discourages or hinders contact with Eco AspRS, being either a neutral element or an antideterminant respectively. This base-pair also has an impact on aspartylation by Eco AspRS in the Eco tRNAAsp framework, leading to a 4-fold global loss in aminoacylation efficiency (small positive effect on kcat and a 6-fold increase of KM, Table 2). In summary, Eco AspRS is sensitive to the nucleotides present in base-pair 11e24 in either of the two tRNA frameworks tested. The individual contributions of base-pair 11e24 and of D/T-loop interaction to aspartylation efficiency of the mt-tRNA appears to be additive [30]. Indeed, whatever the order of insertion of these two features into mt-tRNAAspidec, the final gain in aminoacylation efficiency is w28 fold (Fig. 1). The contribution of the loop architecture is a 2.5- to 4-fold gain and that of base-pair U11eA24 is a 7- to 11-fold gain in aminoacylation efficiency. Accordingly, conversion of tRNAAspidec into tRNAAspidec/DT/ U11eA24 with tRNAAspidec/U11eA24 as an intermediate variant leads to a cumulative gain of 11-fold and 2.5-fold, i.e. a total of 27.5fold, and with tRNAAspidec/DT as the intermediate leads to a gain of 4-fold and 7-fold, i.e. a total gain of 28-fold. The contribution of base-pair 11e24 to aspartylation by Eco AspRS remains dependent on additional elements of the framework, however, as illustrated by the variability in aminoacylation levels of four mammalian mt-tRNAAsp variants, transplanted with the complete Eco aspartate identity elements (Fig. 2). The absence
of base-pair U11eA24 in mt-tRNAAsp from Lemur catta, Mus musculus or Ornithorhynchus anatinus, or its presence in Pan troglodytes mt-tRNAAsp, is not correlated with the charging level (Fig. 2). Accordingly, the framework itself is important for aminoacylation of tRNA substrates by Eco AspRS. 3.5. Large gap in conformational plasticity between mt- and Eco tRNAs An important parameter contributing to the interaction between a tRNA and an aaRS is the dynamics required for reciprocal adaptation of both macromolecules [24,31,32]. Furthermore, the aminoacylation reaction itself is triggered by signal transmission from identity elements (often concentrated in the tRNA anticodon loop) to the catalytic site of the enzyme located at more than 70 Å distance, either through the tRNA, the enzyme, or both. The communication pathways are being progressively deciphered using approaches such as comparative crystallography and molecular dynamics simulations (e.g. [33,34]). Access to crystallographic structures of Eco AspRS, both free of substrates and in complex with tRNA, ATP and aspartic acid, revealed both limited global adaptability of the enzyme to its substrates as well as predefined pockets for “lock- and key-type” interactions [35]. Global adaptation takes place upon tRNA binding with rotation of 10e15 of the N-terminal and insertion domains. Predefined and rigid conformations concern two important sites, the region of contact with the tRNA anticodon and the catalytic site. Accordingly, an Eco AspRS tRNA substrate needs to be capable of fitting to these predefined and distant pockets, a property that may rely on stringently controlled structural rigidity of the RNA. The structural properties of mt-tRNAs including enlarged flexibility may not allow this prerequisite to be fulfilled. An estimate of the thermodynamic structural stability/instability of tRNAs can be obtained from calculations of the free energies of their cloverleaf structures. Fig. 3 allows for comparison of the stabilities of the different tRNAs investigated herein as
Fig. 2. Transplantation of the full set of Eco aspartate identity elements is not in itself sufficient to restore aminoacylation levels of mitochondrial tRNAAsp from various mammals. Four tRNAs were evaluated as indicated. Transplanted nucleotides are indicated by arrows. Eco aspartate identity elements are on grey background. Variability in sequence of basepair 11e24 is boxed as a reminder that A11eU24 is of importance for Eco AspRS in the human mt-tRNAAsp background (see Fig. 1 and Table 2) and U11eA24 is the natural sequence in Eco tRNAAsp. Maximum aspartylation levels (percentage of aminoacylated tRNA from the total input population) by Eco AspRS are indicated (tested as described under material and methods). Aminoacylation of reference wild-type tRNA transcripts is not detectable under these conditions.
A. Fender et al. / Biochimie 94 (2012) 1090e1097
1095
overcome the 18 kcal/mol gap in free energy, corresponding to almost total replacement of the initial mt-sequence. 4. Conclusion and perspectives
Fig. 3. Structural stability of Eco tRNAAsp and Hsa mt-tRNAAsp. Free energies were calculated with the RNAeval program of the Vienna RNA package, with fixed secondary structure cloverleaf constraints and for a temperature of 15 C. Wild-type and transplanted mammalian mitochondrial tRNAAsp variants cloverleaf structures are approximately 2-fold less stable than Eco tRNAAsp (wild-type and variants).
calculated with the RNAeval program of the Vienna RNA package. The calculations do not take into account the D-/T-loop interactions nor any tertiary interaction and are performed on unmodified transcript sequences; they highlight impressive differences between the stability of Eco tRNA derivatives (about 42 kcal/mol) and mt-tRNA variants (about 24 kcal/mol) at 15 C (see free energies at different temperatures in Supplementary Table 1), however, supporting a far higher stability of Eco tRNAs than mttRNAs. These calculations are also in agreement with large differences in melting temperatures measured for in vitro transcribed “classical” tRNAs (about 60 C for yeast tRNAPhe [36]; and yeast tRNAAsp [20]) and Hsa mt-tRNA (e.g. 37 C for tRNALys [37]). A small population of Hsa mt-tRNAAsp also folds into alternative structures such as an incomplete cloverleaf and an extended hairpin [23]. The free energies of these structures are even weaker than that of the cloverleaf fold, with DG ¼ 18 kcal/mol and 13 kcal/mol respectively. We propose that the about two-fold lower stability of the mttRNAs as compared to Eco tRNAAsp represents a strong barrier for proper accommodation to the Eco AspRS. This enzyme is probably unable to deal properly with the too large intrinsic flexibility of the mt-tRNA substrate. A variant was prepared in which base-pair U31eA39 in the anticodon stem was replaced by C31eG39 within the most efficient mt-tRNAAsp derivative constructed so far, namely mt-tRNAAspidec/ DT/U11eA24 (L ¼ 260-fold). Base-pair 31e39 was chosen since a similar substitution, A31$C39 to G31eC39 in Hsa mt-tRNALeu(UUR), was previously shown to convert an otherwise unfolded domain into a helical folding of both anticodon and D-stems [38,39]. The resulting variant mt-tRNAAspidec/DT/U11eA24/C31eG39 is chargeable by both synthetases (data not shown) and indeed, causes a 10-fold increase in the catalytic rate constant of aspartylation by Eco AspRS (Table 2). This positive effect is due to a 35-fold increase in KM so that the final aspartylation efficiency remains 635-fold lower than for wt Eco tRNAAsp. A systematic search for individual or combined base-pairs of Hsa mt-tRNAAspidec/DT/ U11eA24 to be converted from weak AeU or GeU pairs into more stable GeC pairs, would likely make possible the full conversion of the mt-tRNA into an optimal substrate for the Eco AspRS. However, this would require conversion of about 9 pairs to
The case study presented herein pinpoints a number of molecular reasons for the cross-aminoacylation barrier between a mammalian mt-tRNA and a bacterial aaRS. As a result we propose that the absence of aminoacylation of Hsa mt-tRNAAsp by Eco AspRS is linked to three major drawbacks: (i) incompleteness of features defining classical aminoacylation rules (including identity elements and a structural motif), (ii) the presence of at least one idiosyncratic antideterminant or neutral element and possible additional context elements, and (iii) too large intrinsic and global conformational flexibility hindering optimal adaptation to the synthetase. Accordingly, and as opposed to other cross-aminoacylation barriers discussed in the Introduction and dealing with tRNAs from various kingdoms and various species but all of same “classical” structural organization, conversion of a mt-tRNA into an efficient substrate for a bacterial synthetase cannot be solved uniquely by considering general aminoacylation rules but is also based on thermodynamic properties of the tRNA. The present data have been obtained on in vitro transcribed tRNAs, deprived of post-transcriptional modifications. Posttranscriptional modifications are known to restrict the landscape of alternative structures of tRNAs (as demonstrated in the case of Hsa mt-tRNALys [40,41]) and to stabilize RNA conformations (e.g. [20,36]). Native Hsa mt-tRNAAsp has only 4 modifications (m1A9, m2G10, J27, Q34 [23]) and native Eco tRNAAsp has 9 modifications (s4U8, D16, D20, D20:1, Q34, m2A37, m7G46, T54, J55; [42]). Even if not all modifications lead to stabilization (e.g. s4U or D increase local flexibility), it is likely that while both native tRNAs fold into the same structure, they can still be distinguished by differences in stability (i.e., the Eco native tRNAAsp being more stable than the native mt-tRNAAsp) and that the general conclusions derived above likely remain valid. The fact that variants derived from mt-tRNAAsp coming from 4 additional mammals are aminoacylated to different extents further suggests that detailed idiosyncratic structural features also contribute to the cross-aminoacylation barrier with Eco AspRS. Base-pair 11e24 is one of these. It is interesting to note that this base-pair, investigated as the “Hirsch suppressor” when mutated in the Eco tRNATrp, was recently shown to act through an increase in flexibility on the ribosome [43]. The contribution of the tRNA partner to successful induced-fit to the synthetase has been reported previously (e.g. [44]). In the case of mt-tRNAs, however, this is amplified in an unprecedented way. Mt-tRNAs are coded by both strands of the mammalian mtgenome which are of highly different nucleotide composition (Grich or “heavy” strand; C-rich or “light” strand). In combination with the high mutation rate of the mt-DNA, the population of mttRNAs covers a large range of structural diversity, with “light” and “heavy” tRNAs in terms of nucleotide content, and with “classical” to “highly degenerated” tRNAs in regard of 3D structures and stability [22,24]. It can thus be anticipated that the barriers for cross-aminoacylation of the set of “light” tRNAs by bacterial enzymes would display the same general trend as those deciphered for mt-tRNAAsp. Restriction of the set of identity elements within mammalian mt-tRNAs has been observed for other aminoacylation systems investigated so far, namely those for tyrosine [45] and serine [46,47]. Furthermore, all tRNAs considered in the pioneering work of Kumazawa and colleagues (Table 1, [7,8]) are “light” tRNAs that share the structural properties of Hsa tRNAAsp. While the present work highlights peculiar tRNA properties that contribute to restricted recognition by aaRSs, an intriguing unsolved question concerns the properties of the bacterial and
1096
A. Fender et al. / Biochimie 94 (2012) 1090e1097
mitochondrial synthetases responsible for their different behaviours when facing the same tRNA substrate. Initial answers to this question come from genetic approaches which allow conversion of a bacterial synthetase into a yeast mitochondrial enzyme [48,49]. A fine-tuned comparative analysis of both Hsa mt-AspRS and Eco AspRS has already shown how the mt-AspRS deals with the absence of the major aspartate identity element G73, revealing subtle local evolutionary adaptation by a point mutation in the binding pocket [14]. Full understanding requires further explorations and in particular access to crystallographic structures and refined thermodynamic investigations of tRNA/synthetase partnerships. Work is in progress along these lines.
Acknowledgements We thank R. Giegé and L. Levinger for critical reading and helpful comments on the manuscript, G. Eriani, H.D. Becker and D. Kern for gift of material. This work was supported by Centre National de la Recherche Scientifique, Université de Strasbourg, Association Française contre les Myopathies and Action Concertée Incitative (ACI-BCMS 146). A.F. was supported by a fellowship from Ministère de l’Enseignement Supérieur et de la Recherche and F.J. by the German Academic Exchange Service (DAAD D/10/43622).
Appendix. Supplementary material Supplementary data associated with this article can be found, in the online version, at 10.1016/j.biochi.2012.02.030.
References [1] R. Giegé, M. Sissler, C. Florentz, Universal rules and idiosyncratic features in tRNA identity, Nucleic Acids Res. 26 (1998) 5017e5035. [2] P.J. Beuning, K. Musier-Forsyth, Transfer RNA recognition by aminoacyl-tRNA synthetases, Biopolymers 52 (1999) 1e28. [3] R. Giegé, M. Frugier, Transfer RNA structure and identity, in: J. Lapointe, L. Brakier-Gringas (Eds.), Translation Mechanisms, Landes Sciences, Georgetown, TX, 2003, pp. 1e24. [4] R. Giegé, B. Rees, Aspartyl-tRNA synthetases, in: M. Ibba, C. Francklyn, S. Cusack (Eds.), Aminoacyl-tRNA Synthetases, Landes Bioscience, Georgetown, TX, 2005, pp. 210e226. [5] M.E. Saks, J.R. Sampson, Variant minihelix RNAs reveal sequence-specific recognition of the helical tRNASer acceptor stem by E. coli seryl-tRNA synthetase, EMBO J. 15 (1996) 2843e2849. [6] M. Frugier, M. Helm, B. Felden, R. Giegé, C. Florentz, Sequences outside recognition sets are not neutral for tRNA aminoacylation: evidence for nonpermissive combinations of nucleotides in the acceptor stem of yeast tRNAPhe, J. Biol. Chem. 273 (1998) 11605e11610. [7] Y. Kumazawa, T. Yokogawa, E. Hasegawa, K.-I. Miura, K. Watanabe, The aminoacylation of structurally variant phenylalanine tRNAs from mitochondria and various nonmitochondrial sources by bovine mitochondrial phenylalanyltRNA synthetase, J. Biol. Chem. 264 (1989) 13005e13011. [8] Y. Kumazawa, H. Himeno, K.-I. Miura, K. Watanabe, Unilateral aminoacylation specificity between bovine mitochondria and eubacteria, J. Biochem. 109 (1991) 421e427. [9] B. Brindefalk, J. Viklund, D. Larsson, M. Thollesson, S.G. Andersson, Origin and evolution of the mitochondrial aminoacyl-tRNA synthetases, Mol. Biol. Evol. 24 (2007) 743e756. [10] L. Bonnefond, A. Fender, J. Rudinger-Thirion, R. Giegé, C. Florentz, M. Sissler, Towards the full set of human mitochondrial aminoacyl-tRNA synthetases: characterization of AspRS and TyrRS, Biochemistry 44 (2005) 4805e4816. [11] M. Sissler, J. Pütz, F. Fasiolo, C. Florentz, Mitochondrial aminoacyl-tRNA synthetases, in: M. Ibba, C. Francklyn, S. Cusack (Eds.), Aminoacyl-tRNA Synthetases, Landes Biosciences, Georgetown, TX, 2005, pp. 271e284. [12] G. Eriani, M. Delarue, O. Poch, J. Gangloff, D. Moras, Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs, Nature 347 (1990) 203e206. [13] S. Cusack, C. Berthet-Colominas, M. Härtlein, N. Nassar, R. Leberman, A second class of synthetase structure revealed by X-ray analysis of Escherichia coli seryl-tRNA synthetase at 2.5 Å, Nature 347 (1990) 249e255. [14] A. Fender, C. Sauter, M. Messmer, J. Pütz, R. Giegé, C. Florentz, M. Sissler, Loss of a primordial identity element for a mammalian mitochondrial aminoacylation system, J. Biol. Chem. 281 (2006) 15980e15986.
[15] J. Pütz, J.D. Puglisi, C. Florentz, R. Giegé, Identity elements for specific aminoacylation of yeast tRNAAsp by cognate aspartyl-tRNA synthetase, Science 252 (1991) 1696e1699. [16] H.D. Becker, R. Giegé, D. Kern, Identity of prokaryotic and eukaryotic tRNAAsp for aminoacylation by aspartyl-tRNA synthetase from Thermus thermophilus, Biochemistry 35 (1996) 7447e7458. [17] H.D. Becker, J. Reinbolt, R. Kreutzer, R. Giegé, D. Kern, Existence of two distinct aspartyl-tRNA synthetases in Thermus thermophilus. Structural and biochemical properties of the two enzymes, Biochemistry 36 (1997) 8785e8797. [18] T. Hasegawa, H. Himeno, H. Ishikura, M. Shimizu, Discriminator base of tRNAAsp is involved in amino acid acceptor activity, Biochem. Biophys. Res. Commun. 163 (1989) 1534e1538. [19] N. Nameki, K. Tamura, H. Himeno, H. Asahara, T. Hasegawa, M. Shimuzu, E. coli tRNAAsp recognition mechanism differing from that of the yeast system, Biochem. Biophys. Res. Commun. 189 (1992) 856e862. [20] V. Perret, A. Garcia, J.D. Puglisi, H. Grosjean, J.-P. Ebel, C. Florentz, R. Giegé, Conformation in solution of yeast tRNAAsp transcripts deprived of modified nucleotides, Biochimie 72 (1990) 735e744. [21] P. Fechter, J. Rudinger, R. Giegé, A. Théobald-Dietrich, Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity, FEBS Lett. 436 (1998) 99e103. [22] M. Helm, H. Brulé, D. Friede, R. Giegé, J. Pütz, C. Florentz, Search for characteristic structural features of mammalian mitochondrial tRNAs, RNA 6 (2000) 1356e1379. [23] M. Messmer, J. Pütz, T. Suzuki, T. Suzuki, C. Sauter, M. Sissler, C. Florentz, Tertiary network in mammalian mitochondrial tRNAAsp revealed by solution probing and phylogeny, Nucleic Acids Res. 37 (2009) 6881e6895. [24] R. Giegé, F. Jühling, J. Pütz, P. Stadler, C. Sauter, C. Florentz, Structure of transfer RNAs: similarity and variability, Wiley Interdiscip. Rev. RNA 3 (2012) 37e61. [25] M. Frugier, C. Florentz, R. Giegé, Efficient aminoacylation of resected RNA helices by class II aspartyl-tRNA synthetase dependent on a single nucleotide, EMBO J. 13 (1994) 2218e2226. [26] H. Choi, G. Kay, J.A. Schneider, S. Otten, W.H. McClain, Recognition of acceptorstem structure of tRNAAsp by Escherichia coli aspartyl-tRNA synthetase, RNA 9 (2003) 386e393. [27] J. Pütz, B. Dupuis, M. Sissler, C. Florentz, Mamit-tRNA, a database of mammalian mitochondrial tRNA primary and secondary structures, RNA 13 (2007) 1184e1190. [28] G. Dirheimer, G. Keith, P. Dumas, E. Westhof, Primary, secondary and tertiary structures of tRNAs, in: D. Söll, U.L. RajBhandary (Eds.), tRNA: Structure, Biosynthesis, and Function, Am. Soc. Microbiol. Press, Washington, DC, 1995, pp. 93e126. [29] S. Eiler, A.C. Dock-Bregeon, L. Moulinier, J.-C. Thierry, D. Moras, Synthesis of aspartyl-tRNAAsp in Escherichia coli-a snapshot of the second step, EMBO J. 18 (1999) 6532e6541. [30] J. Pütz, J.D. Puglisi, C. Florentz, R. Giegé, Additive, cooperative and anticooperative effects between identity nucleotides of a tRNA, EMBO J. 12 (1993) 2949e2957. [31] S. Hauenstein, C.M. Zhang, Y.M. Hou, J.J. Perona, Shape-selective RNA recognition by cysteinyl-tRNA synthetase, Nat. Struct. Mol. Biol. 11 (2004) 1134e1141. [32] R. Giegé, Toward a more complete view of tRNA biology, Nat. Struct. Mol. Biol. 15 (2008) 1007e1014. [33] A. Sethi, J. Eargle, A.A. Black, Z. Luthey-Schulten, Dynamical networks in tRNA:protein complexes, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 6620e6625. [34] R.W. Alexander, J. Eargle, Z. Luthey-Schulten, Experimental and computational determination of tRNA dynamics, FEBS Lett. 584 (2010) 376e386. [35] B. Rees, G. Webster, M. Delarue, M. Boeglin, D. Moras, Aspartyl-tRNA synthetase from Escherichia coli: flexibility and adaptability to the substrates, J. Mol. Biol. 299 (2000) 1157e1164. [36] J.R. Sampson, O.C. Uhlenbeck, Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 1033e1037. [37] C. Florentz, H. Brulé, M. Helm, R. Giegé, Structural studies of human mitochondrial tRNAs, in: P. Lestienne (Ed.), Mitochondrial Diseases, Springer Verlag, Heidelberg, 1999, pp. 59e72. [38] B. Sohm, M. Sissler, H. Park, M.P. King, C. Florentz, Recognition of human mitochondrial tRNALeu(UUR) by its cognate leucyl-tRNA synthetase, J. Mol. Biol. 339 (2004) 17e29. [39] E.I. Zagryadskaya, S.O. Kelley, Combinatorial analysis of loop nucleotides in human mitochondrial tRNALeu(UUR), Biochemistry 44 (2005) 233e242. [40] M. Helm, H. Brulé, F. Degoul, C. Cepanec, J.-P. Leroux, R. Giegé, C. Florentz, The presence of modified nucleotides is required for cloverleaf folding of a human mitochondrial tRNA, Nucleic Acids Res. 26 (1998) 1636e1643. [41] Y. Motorin, M. Helm, tRNA stabilization by modified nucleotides, Biochemistry 49 (2010) 4934e4944. [42] F. Jühling, M. Mörl, R.K. Hartmann, M. Sprinzl, P.F. Stadler, J. Pütz, tRNAdb 2009: compilation of tRNA sequences and tRNA genes, Nucleic Acids Res. 37 (2009) D159eD162. [43] X. Agirrezabala, E. Schreiner, L.G. Trabuco, J. Lei, R.F. Ortiz-Meoz, K. Schulten, R. Green, J. Frank, Structural insights into cognate versus near-cognate discrimination during decoding, EMBO J. 30 (2011) 1497e1507.
A. Fender et al. / Biochimie 94 (2012) 1090e1097 [44] L. Moulinier, S. Eiler, G. Eriani, J. Gangloff, J.-C. Thierry, K. Gabriel, W.H. McClain, D. Moras, The structure of an AspRS-tRNAAsp complex reveals a tRNA-dependant control mechanism, EMBO J. 20 (2001) 5290e5301. [45] L. Bonnefond, M. Frugier, R. Giegé, J. Rudinger-Thirion, Human mitochondrial TyrRS disobeys the tyrosine idenity rules, RNA 11 (2005) 558e562. [46] T. Ueda, Y. Yotsumoto, K. Ikeda, K. Watanabe, The T-loop region of animal mitochondrial tRNASer(AGY) is a main recognition site for homologous seryltRNA synthetase, Nucleic Acids Res. 20 (1992) 2217e2222.
1097
[47] S. Chimnaronk, M. Gravers Jeppesen, T. Suzuki, J. Nyborg, K. Watanabe, Dualmode recognition of noncanonical tRNASer by seryl-tRNA synthetase in mammalian mitochondria, EMBO J. 24 (2005) 3369e3379. [48] H.Y. Huang, Y. Kuei, H.Y. Chao, S.J. Chen, L.S. Yeh, C.C. Wang, Cross-species and cross-compartmental aminoacylation of isoaccepting tRNAs by a class II tRNA synthetase, J. Biol. Chem. 281 (2006) 31430e31439. [49] W.C. Chiu, C.P. Chang, C.C. Wang, Evolutionary basis of converting a bacterial tRNA synthetase into a yeast cytoplasmic or mitochondrial enzyme, J. Biol. Chem. 284 (2009) 23954e23960.
Contents lists available at SciVerse ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
Adaptation of aminoacylation identity rules to mammalian mitochondria Aurélie Fender, Agnès Gaudry, Frank Jühling, Marie Sissler, Catherine Florentz* Architecture et Réactivité de l’ARN, CNRS, Université de Strasbourg, IBMC, 15 rue René Descartes, F-67084 Strasbourg Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 January 2012 Accepted 23 February 2012 Available online 2 March 2012
Many mammalian mitochondrial aminoacyl-tRNA synthetases are of bacterial-type and share structural domains with homologous bacterial enzymes of the same specificity. Despite this high similarity, synthetases from bacteria are known for their inability to aminoacylate mitochondrial tRNAs, while mitochondrial enzymes do aminoacylate bacterial tRNAs. Here, the reasons for non-aminoacylation by a bacterial enzyme of a mitochondrial tRNA have been explored. A mutagenic analysis performed on in vitro transcribed human mitochondrial tRNAAsp variants tested for their ability to become aspartylated by Escherichia coli aspartyl-tRNA synthetase, reveals that full conversion cannot be achieved on the basis of the currently established tRNA/synthetase recognition rules. Integration of the full set of aspartylation identity elements and stabilization of the structural tRNA scaffold by restoration of D- and T-loop interactions, enable only a partial gain in aspartylation efficiency. The sequence context and high structural instability of the mitochondrial tRNA are additional features hindering optimal adaptation of the tRNA to the bacterial enzyme. Our data support the hypothesis that non-aminoacylation of mitochondrial tRNAs by bacterial synthetases is linked to the large sequence and structural relaxation of the organelle encoded tRNAs, itself a consequence of the high rate of mitochondrial genome divergence. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Identity elements Mitochondrial tRNA Cross-aminoacylation Structural plasticity
1. Introduction Rules governing interaction of tRNAs and aminoacyl-tRNA synthetases (aaRSs1) for efficient and specific aminoacylation have been well established for a large number of aminoacylation systems in the three kingdoms of life (for reviews, see [1e4]). They rely on identity nucleotides, mainly located at both extremities of the tRNA L-shaped 3D structure that contact specific amino acids in the aaRS structure. Identity sets are composed of major and minor elements, with major elements usually conserved during evolution, as is the architecture of tRNA. Accordingly, it became possible to convert the specificity of a given tRNA by simple transplantation of identity sets, and/or by removal of antideterminants (signals hindering recognition by a synthetase of other specificity). Cryptic signals and permissive nucleotides that also contribute to specificity were discovered in this context [5,6]. Cross-species aminoacylation of tRNAs from a given organism by synthetases of the same specificity but from other organisms were reported in great numbers [1]. Exceptions where cross-aminoacylation was not
* Corresponding author. Tel.: þ33 (0) 3 88 41 70 59; fax: þ33 (0) 3 88 60 22 18. E-mail address: [email protected] (C. Florentz). 1 aaRS stands for aminoacyl-tRNA synthetase with aa for amino acid (for individual aaRS, aa is given in the 3 letter code, e.g. AspRS for aspartyl-tRNA synthetase). 0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2012.02.030
possible were explained by the presence of only partial sets of identity elements in the tRNA or by the existence of alternate sets, and in many cases could be overcome by simple mutagenic approaches in vitro [1]. An unsolved cross-aminoacylation barrier concerns mammalian mitochondrial (mt) tRNAs which are not aminoacylated by bacterial synthetases [7,8]. In mammalian mitochondria, aminoacylation systems are of dual origin with the tRNAs coded by a simplified and rapidly evolving mt-genome and aaRSs coded by the slowly evolving nuclear genome and imported into mitochondria. Mammalian mtaaRSs have various evolutionary origins [9] but many are of bacterial-type ([10,11] and references therein) and do not depart from the already known synthetases. The human mt-aaRSs (Hsa mtaaRS) have no drastic apparent structural peculiarities as compared to their evolutionarily-related bacterial counterparts (except PheRS, which is a monomer instead of an a2b2 tetramer), i.e. they have the same structural organization, possess the expected class-specific signature motifs in their catalytic domains and, accordingly, belong to the expected aaRS-class [12,13]. However, Kumazawa and colleagues [7,8] reported already twenty years ago on aminoacylation of Escherichia coli (Eco) and Thermus thermophilus (Tth) tRNAs by bovine mt-aaRSs specific for threonine, arginine, lysine, phenylalanine and serine, but absence of charging of bovine (Bos taurus or Bta) mt-tRNAs by the corresponding bacterial aaRSs (Table 1). This is intriguing since three of these enzymes (Bta mt-
A. Fender et al. / Biochimie 94 (2012) 1090e1097
2.2. tRNA gene mutagenesis and in vitro transcription
Table 1 Unilateral aminoacylation of mammalian mitochondrial tRNAs. aa
tRNA
aaRS Bta mt
Eco
Tth
Phe
Bta mt Eco Tth Bta mt Eco Tth Bta mt Eco Tth Bta mt Eco Tth Bta mt Eco Tth
YES YES YES YES YES YES YES YES WEAK YES Hsa mt YES YES
NO þ NO þ WEAK þ NO þ NO þ Eco NO nt
NO þ NO þ NO þ NO NO þ Tth NO þ
Thr
Arg
Lys
Ser
Asp
Hsa mt Eco Tth
1091
Ref
[7]
[8]
[8]
[8]
[8]
[14]
Only aminoacylation capacities for couples involving a single mitochondrial partner, either mitochondrial tRNA or mitochondrial aminoacyl-tRNA synthetase, are emphasized by YES (active), NO (inactive) or WEAK (partially active). Aminoacylation capacities of other couples are indicated as follows: positive control activities of cognate couples (); aminoacylation activity for systems involving both bacterial tRNA and bacterial synthetase (þ/); non tested (nt). Bta mt: Bos taurus mitochondrial; Hsa mt: Homo sapiens mitochondrial; Eco: Escherichia coli; Tth: Thermus thermophilus.
ThrRS, -PheRS and -SerRS) are of the bacterial-type [9,11]. A similar barrier was observed for the Hsa mt-aspartylation system from which the tRNA is not recognized by a bacterial synthetase, i.e. either Eco AspRS or Tth AspRS, while the Hsa mt-AspRS, which is of prokaryotic-type, recognizes and aminoacylates both Eco and Tth tRNAAsp ( [14]; our unpublished results) (Table 1). These data demonstrate unilateral aminoacylation of mt-tRNAs by mt-aaRSs, and show a strong cross-species barrier for aminoacylation of mttRNAs by bacterial synthetases. To sort out the possible molecular reasons for this barrier, the specific case of non-aminoacylation of Hsa mt-tRNAAsp by the Eco AspRS was investigated as a model system by a mutagenic in vitro approach. As a result, we have observed that the absence of classical aminoacylation signals, namely identity and structural elements, are key elements for non-cross-aminoacylation of the mt-tRNA, but that the intrinsic unusually large thermodynamically-driven structural plasticity of the mt-tRNA also forms an important element in the non-recognition barrier. We propose that nonaminoacylation of mammalian mt-tRNAs by bacterial synthetases is linked to the global divergence of the organelle encoded tRNAs, far beyond loss of identity elements and well-defined structural features, a consequence of the very high mutation rate of mitochondrial genomes. 2. Material and methods
Hsa mt-tRNAAsp variants displaying individual Eco identity elements [18,19] (mt-tRNAAspG73, mt-tRNAAspC38 and mttRNAAspG2eC71) or “classical” D- and T-loops as found in tRNAAsp from Schizosaccharomyces pombe (mt-tRNAAspDT) have been prepared previously [14]. Hsa mt-tRNAAsp variant (G73/DT, C38/G73, idec,2 idec/DT, idec/U11eA24, and idec/DT/U11eA24) genes were obtained by hybridization of 9 overlapping oligonucleotides, ligation between BamHI and HindIII sites of plasmid pTFMa [20]. The gene for variant idec/DT/U11eA24/C31eG39 was derived from the idec/DT/ U11eA24 gene with the QuikChangeÔ Site-Directed Mutagenesis Kit (Stratagene). All these genes contain a hammerhead ribozyme [21] and tRNA sequences downstream from the T7 polymerase promoter. A BstNI site coincidental with the 30 -end of the tRNA sequences allows synthesis of tRNAs ending with the expected CCA sequence. Eco tRNAAsp variant (mtDT and A11eU24) genes were obtained by PCR amplification using two complementary 30 nt-long primers (allowing for introduction of BamHI and HindIII sites) that contain the T7 polymerase promoter and tRNA sequences. All synthetic genes were transformed into TOP10 cells (Invitrogen). Transcription was as described [21]. tRNAs were purified to single nucleotide resolution on denaturing polyacrylamide gels as described [14]. Quality of tRNAs has been verified on native PAGE which revealed only single populations of conformers. 2.3. E. coli and human mt-AspRS Eco AspRS was a gift from G. Eriani (IBMC, Strasbourg). Hsa mtAspRS was expressed and purified on a nickel affinity column [10]. Protein concentrations were determined from OD280 using the extinction coefficients (ε(Eco$AspRS) ¼ 42985 M1 cm1 and ε(Hsa$mt1 cm1) and molecular weights (MW(Eco$AspRS) AspRS) ¼ 43540 M ¼ 65913.4 g mol1 and MW(Hsa$mt-AspRS) ¼ 69627 g mol1) calculated with ProtParam from ExPASy tools (expasy.org). 2.4. Aminoacylation of tRNAs Aminoacylation assays were performed as described [14]. Assays were conducted at 15 C in 50 mM HEPES-KOH pH 7.5, 25 mM KCl, 12 mM MgCl2, 2.5 mM ATP, 0.2 mg/mL BSA, 1 mM spermine, 32 mM [3H]-aspartic acid (208 GBq/mmol), and adequate amounts of transcripts and aaRS. Assays were performed in 50 mL samples. Transcripts were renatured at 60 C (for mt-tRNAAsp and variants) or 85 C (for Eco tRNAAsp and variants) for 90 s in water and slow cooling down to room temperature before aminoacylation. Maximal charging levels (plateaus) were determined for 400 nM transcript and optimal concentrations of synthetase (500 nM Hsa mt-AspRS or 200 nM Eco AspRS). Kinetic parameters kcat and KM were derived from LineweavereBurk plots obtained using a range of tRNA concentration from 200 nM to 8 mM (the very high KM of 70 mM for one variant is an approximation) and of Eco AspRS from 2 to 100 nM. Experiments were performed at least in triplicate on separate preparations of transcripts. Final kcat and KM values varied up to 40%.
2.1. Materials Synthetic genes of wild-type Eco tRNAAsp [15] and Hsa mttRNAAsp [10] were cloned previously. T7 RNA polymerase was purified as described [16]. Tth AspRS (discriminating version [17]) and the corresponding in vitro transcribed tRNAAsp were gifts from H.D. Becker and D. Kern (IBMC, Strasbourg). Oligonucleotides were from Sigma Genosys, restriction enzymes (BamHI, HindIII, and BstNI) from New England Biolabs, T4 DNA ligase from Qbiogen, and 3 L-[ H] aspartic acid (208 GBq/mmol) from Amersham.
2.5. Thermodynamic stability of tRNAs Free energies (expressed as kcal/mol) of tRNA structures were calculated with the RNAeval program of the Vienna RNA package (http://rna.tbi.univie.ac.at/cgi-bin/RNAeval.cgi), with fixed secondary
2 Idec stands for identity elements of Eco tRNAAsp for aspartylation by Eco AspRS, namely G73, G2eC71, G10, G34, U35, C36, C38.
1092
A. Fender et al. / Biochimie 94 (2012) 1090e1097
structure constraints and with a temperature of 15 C. Advanced options “dangling energies on both sides of a helix in any case” and “RNA parameters (Turner Model, 2004)” were applied. 3. Results and discussion 3.1. Characteristic features of heterologous Hsa mt-tRNAAsp and Eco AspRS Hsa mt-AspRS is of the prokaryotic-type, with the typical bacterial insertion domain and C-terminal extension, and shares 43% of strictly identical residues with Eco AspRS [10]. The human mt-AspRS possesses residues strictly conserved in all known AspRS sequences which include residues involved in ATP-, tRNA-, and amino acid-binding (those typical for class II aaRSs and those specific for aspartic acid recognition) [10]. While the two enzymes are similar, the corresponding tRNAs are markedly different. Hsa mt-tRNAAsp (Fig. 1) is a typical example of mammalian mt-tRNA in terms of sequence and structural features [22] and is to a large extent distinct from Eco tRNAAsp (Fig. 1). It has a highly skewed A, U and C nucleotide content (90% versus 66%), a high content of weak AeU and GeU base-pairs (16 out of 21 versus 7 out of 21) and nonclassical D- and T-loops. In terms of 3D structures, both tRNAs are however of the same type, with a common network of core interactions but with a major difference in the elbow of the L-shape where D/T-loop interactions stabilize the Eco tRNAAsp but are absent in the mt-tRNAAsp [23]. The two tRNAs are typical structural
representatives of bacterial tRNAs and mammalian mt-tRNAs, respectively [24]. In terms of signals responsible for aspartate aminoacylation specificity, Eco tRNAAsp possesses the major aspartate determinants identified in all “classical” (i.e. non organelle) aspartate systems investigated so far (reviewed in [1,4]), namely the discriminator residue G73 (a primordial element, sufficient for specific aminoacylation of a minihelix mimicking the acceptor branch of an L-shaped tRNA, [25]), the anticodon triplet G34U35C36 and base-pair G10eC25 (U25 is also tolerated). Eco AspRS is also sensitive to additional minor elements, namely residue C38 and base-pair G2eC71 [19,26]. By contrast, in the Hsa mt-tRNAAsp, the aspartate identity set is restricted to the anticodon triplet G34U35C36 where only U35 and G36 are major determinants [14]. This minimal set is sufficient for specific aspartylation by Hsa mt-AspRS. Notably, the discriminator residue 73 and minor elements such as base-pair G10eC25 are not involved. The existence of a restricted set of aspartate identity elements is valid all over mammalian mt-tRNAAsp [27]. As a consequence of these properties, a trivial hypothesis for the non-aspartylation of the mt-tRNA by Eco AspRS would be the absence of identity elements G73, G2eC71 and C38, and the absence of a structural determinant that would provide a stable D/T-loop interaction, as suggested previously for the cases presented in Table 1 [8]. In what follows, the contribution of these elements has been investigated by a mutagenic approach aimed at progressively converting mt-tRNAAsp into an efficient substrate for aspartylation by Eco AspRS.
Fig. 1. Attempts to convert Hsa mt-tRNAAsp into an efficient substrate for Eco AspRS through application of classical aminoacylation identity rules. A. Variants of Hsa mt-tRNAAsp highlighting progressive incorporation of bacterial tRNAAsp signature motifs. Newly inserted mutations into the global background of human mt-tRNAAsp (grey line) are explicitly indicated; “idec” refers to the identity elements of Eco tRNAAsp. The aminoacylation efficiency by Eco AspRS is indicated (L for loss in aminoacylation efficiency as compared to Eco tRNAAsp for which L ¼ 1) as well as the incidence of the mutations on individual kinetic parameters kcat and KM. G stands for gain in aminoacylation efficiency between two variants. All values are calculated from data presented in Table 2 according to definitions in the legend of Table 2. B. Reference tRNAs, Hsa mt-tRNAAsp wild-type transcript (B1); Eco tRNAAsp wild-type transcript (B2). Eco aspartate identity elements are circled (dark circle for major elements, light grey for minor elements). Tertiary interactions leading to the final Lshaped 3D structures are indicated by dotted lines [24].
A. Fender et al. / Biochimie 94 (2012) 1090e1097
3.2. The full aspartate identity set is necessary but not sufficient for efficient cross-aminoacylation of mt-tRNAAsp In an attempt to convert the human mt-tRNAAsp into a more efficient substrate for Eco AspRS, transcripts with the missing Eco identity elements G73, C38 and base-pair G2eC71 transplanted into them, either individually or in combination, were prepared and tested with both Hsa mt-AspRS and Eco AspRS (Fig. 1). Charging levels are presented in Supplementary Fig. S1, and kinetic parameters in Table 2. While all variants remain fully active for aspartylation by Hsa mt-AspRS as tested previously [14], only the variant hosting the full set of Eco aspartate identity elements, mt-tRNAAspidec (idec for identity element from Eco tRNAAsp) (Fig. 1), became detectably charged by the bacterial enzyme. The other mt-tRNAAsp derivatives, and especially the one including residue G73, remained inactive with charging levels lower than 1%. Establishment of kinetic parameters (Table 2) revealed that kcat is w70-fold lower and KM w100-fold increased as compared to the aminoacylation features of wild-type Eco tRNAAsp transcript. As a consequence, mttRNAAspidec is 7400-fold less efficiently aminoacylated than the wild-type Eco tRNAAsp. According to these data, transplantation of the full set of Eco identity elements is necessary but decisively insufficient for conversion of Hsa mt-tRNAAsp into an efficient substrate for Eco AspRS. 3.3. The full network of tertiary interactions is an additional important but still insufficient feature Most mammalian mt-tRNAs do not display any obvious interaction between their D- and T-loops, allowing great flexibility in the angle between the two branches that form the L-shaped 3D structure [24]. This flexibility could lead to an unusual range of possible distances between the distal extremities of the tRNA, namely the anticodon- and the CCA-end. To evaluate if Eco AspRS is sensitive to this structural feature, classical D- and T-loops (from S. pombe tRNAAsp, which were investigated previously [14]), were transplanted into the initial wild-type mt-tRNAAsp, replacing the smaller and partially degenerated mt-loops, as well as into mttRNAAspidec, with the expectation that they would enable canonical
1093
tertiary interactions and formation of a stabilized elbow of the Lshaped tRNA. According to crystallographic structures, D/T-loop interactions stabilize the angle formed by the acceptor arm and the anticodon arm of the tRNA 3D structure [24]. While the resulting variants mt-tRNAAspDT and mt-tRNAAspidec/DT were as good substrates as the wt mt-tRNAAsp for mt-AspRS [14], mttRNAAspidec/DT (Fig. 1) undergoes a significant improvement as a substrate for Eco AspRS (Supplementary Fig. S1; Table 2). A 4-fold gain in efficiency is observed when compared to mt-tRNAAspidec activity, mainly due to a 4-fold decrease in KM. This modest but significant improvement shows that Eco AspRS not only requires a full aspartate identity set but also is sensitive to the nature of the D- and T-loops of its tRNA substrate. However, the aspartylation capacity of mt-tRNAAspidec/DT remains still 1850-fold less efficient than that of the homologous bacterial tRNAAsp. The importance of the D/T-loop interaction for aspartylation by the Eco enzyme holds true also in the Eco tRNAAsp framework; replacement of Eco tRNAAsp D- and T-loops by the corresponding mt-domains (variant Eco tRNAAspmtDT, Supplementary Fig. S2) leads to a 10-fold decrease in kcat and a 6.5-fold increase in KM for aspartylation with the Eco synthetase. This variant is 70-fold less efficiently aminoacylated by Eco AspRS than wild-type Eco tRNAAsp (Table 2). 3.4. Role of idiosynchratic base-pair A11-U24 in human mt-tRNAAsp Since simple transplantation of identity elements and correction of a structural defect in Hsa mt-tRNAAsp do not allow for full recognition by Eco AspRS, the role of the context elements and especially the presence of antideterminant(s) or neutral elements were considered. A consensus sequence of all tRNAs proven experimentally to be efficient substrates of Eco AspRS in vitro was established and compared to Hsa mt-tRNAAspidec/DT (Supplementary Fig. S3). Base-pair 11e24 has been pinpointed as possible candidate. Hsa mt-tRNAAsp bears A11eU24 while all classical tRNAs have a semi-conserved Y11eR24 pair (Y for pyrimidine, R for purine) [28]. Further, U11 of Eco tRNAAsp makes two hydrogen bonds (one through the base and one through the ribose) with His114 and Asn116 respectively of Eco AspRS (hinge region), as shown in the crystallographic structure of the tRNAAsp/AspRS
Table 2 Kinetic parameters for tRNA aminoacylation by Eco AspRS. tRNA Asp
Wild-type Eco tRNA
Human mitochondrial tRNAAsp variants Wild-type Hsa mt-tRNAAsp Hsa mt-tRNAAspG73 Hsa mt-tRNAAspC38 Hsa mt-tRNAAspG2eC71 Hsa mt-tRNAAspC38/G73 Hsa mt-tRNAAspidec Hsa mt-tRNAAspDT Hsa mt-tRNAAspidec/DT Hsa mt-tRNAAspidec/U11eA24 Hsa mt-tRNAAspidec/DT/U11eA24 Hsa mt-tRNAAspidec/U11-A24/DT/C31eG39 E. coli tRNAAsp variants Eco tRNAAspA73a Eco tRNAAspA38b Eco tRNAAspC2eG72b Eco tRNAAspmtDT Eco tRNAAspA11eU24
kcat (103 s1)
KM (mM)
kcat/KM (103 s1 mM1)
L
2590 320
0.7 0.1
3685
1
nd nd nd nd nd 39 5 nd 34 3 43 14 23 5 319 75
nd nd nd nd nd 69.3 9 nd 17.5 2.5 7.5 3.5 1.6 0.3 55 9
nd nd nd nd nd 0.5 nd 2.0 5.7 14.4 5.8
nd nd nd nd nd 7400 nd 1850 650 260 635
e e e 248 90 3732 340
e e e 4.6 1.8 4.0 2.0
e e e 53.9 933
555 6 5 70 4
In vitro aminoacylation assays were performed as described in “Material and methods” at 15 C. This temperature was found as an optimal compromise between mt-tRNA stability and enzymatic activity of Eco AspRS [14]. nd, not detectable. L stands for relative loss in aminoacylation efficiency considering wt Eco tRNAAsp as the reference (L ¼ (kcat/KM(Eco$tRNA Asp))/(kcat/KM(other tRNA))). a Data from [18]. b Data from [19].
1094
A. Fender et al. / Biochimie 94 (2012) 1090e1097
complex [29]. Therefore, A11eU24 has been replaced by U11eA24 in tRNAAspidec and tRNAAspidec/DT, leading to tRNAAspidec/ U11eA24 and tRNAAspidec/DT/U11eA24 (Fig. 1). Interestingly, both molecules become significantly better substrates for Eco AspRS and remain fully active for Hsa mt-AspRS (Supplementary Fig. S1). tRNAAspidec/U11eA24 and tRNAAspidec/DT/U11eA24 have catalytic efficiencies only 650-fold and 260-fold lower than the wt Eco tRNAAsp, respectively (Table 2). When compared to parental host tRNAs (tRNAAspidec and tRNAAspidec/DT), introduction of the semi-conserved Y11eR24 pair led to improvements in aminoacylation efficiency of 11- and 7-fold, respectively, mainly due to an 11-fold decrease in KM. Thus, in the framework of mttRNAAsp, base-pair A11eU24 discourages or hinders contact with Eco AspRS, being either a neutral element or an antideterminant respectively. This base-pair also has an impact on aspartylation by Eco AspRS in the Eco tRNAAsp framework, leading to a 4-fold global loss in aminoacylation efficiency (small positive effect on kcat and a 6-fold increase of KM, Table 2). In summary, Eco AspRS is sensitive to the nucleotides present in base-pair 11e24 in either of the two tRNA frameworks tested. The individual contributions of base-pair 11e24 and of D/T-loop interaction to aspartylation efficiency of the mt-tRNA appears to be additive [30]. Indeed, whatever the order of insertion of these two features into mt-tRNAAspidec, the final gain in aminoacylation efficiency is w28 fold (Fig. 1). The contribution of the loop architecture is a 2.5- to 4-fold gain and that of base-pair U11eA24 is a 7- to 11-fold gain in aminoacylation efficiency. Accordingly, conversion of tRNAAspidec into tRNAAspidec/DT/ U11eA24 with tRNAAspidec/U11eA24 as an intermediate variant leads to a cumulative gain of 11-fold and 2.5-fold, i.e. a total of 27.5fold, and with tRNAAspidec/DT as the intermediate leads to a gain of 4-fold and 7-fold, i.e. a total gain of 28-fold. The contribution of base-pair 11e24 to aspartylation by Eco AspRS remains dependent on additional elements of the framework, however, as illustrated by the variability in aminoacylation levels of four mammalian mt-tRNAAsp variants, transplanted with the complete Eco aspartate identity elements (Fig. 2). The absence
of base-pair U11eA24 in mt-tRNAAsp from Lemur catta, Mus musculus or Ornithorhynchus anatinus, or its presence in Pan troglodytes mt-tRNAAsp, is not correlated with the charging level (Fig. 2). Accordingly, the framework itself is important for aminoacylation of tRNA substrates by Eco AspRS. 3.5. Large gap in conformational plasticity between mt- and Eco tRNAs An important parameter contributing to the interaction between a tRNA and an aaRS is the dynamics required for reciprocal adaptation of both macromolecules [24,31,32]. Furthermore, the aminoacylation reaction itself is triggered by signal transmission from identity elements (often concentrated in the tRNA anticodon loop) to the catalytic site of the enzyme located at more than 70 Å distance, either through the tRNA, the enzyme, or both. The communication pathways are being progressively deciphered using approaches such as comparative crystallography and molecular dynamics simulations (e.g. [33,34]). Access to crystallographic structures of Eco AspRS, both free of substrates and in complex with tRNA, ATP and aspartic acid, revealed both limited global adaptability of the enzyme to its substrates as well as predefined pockets for “lock- and key-type” interactions [35]. Global adaptation takes place upon tRNA binding with rotation of 10e15 of the N-terminal and insertion domains. Predefined and rigid conformations concern two important sites, the region of contact with the tRNA anticodon and the catalytic site. Accordingly, an Eco AspRS tRNA substrate needs to be capable of fitting to these predefined and distant pockets, a property that may rely on stringently controlled structural rigidity of the RNA. The structural properties of mt-tRNAs including enlarged flexibility may not allow this prerequisite to be fulfilled. An estimate of the thermodynamic structural stability/instability of tRNAs can be obtained from calculations of the free energies of their cloverleaf structures. Fig. 3 allows for comparison of the stabilities of the different tRNAs investigated herein as
Fig. 2. Transplantation of the full set of Eco aspartate identity elements is not in itself sufficient to restore aminoacylation levels of mitochondrial tRNAAsp from various mammals. Four tRNAs were evaluated as indicated. Transplanted nucleotides are indicated by arrows. Eco aspartate identity elements are on grey background. Variability in sequence of basepair 11e24 is boxed as a reminder that A11eU24 is of importance for Eco AspRS in the human mt-tRNAAsp background (see Fig. 1 and Table 2) and U11eA24 is the natural sequence in Eco tRNAAsp. Maximum aspartylation levels (percentage of aminoacylated tRNA from the total input population) by Eco AspRS are indicated (tested as described under material and methods). Aminoacylation of reference wild-type tRNA transcripts is not detectable under these conditions.
A. Fender et al. / Biochimie 94 (2012) 1090e1097
1095
overcome the 18 kcal/mol gap in free energy, corresponding to almost total replacement of the initial mt-sequence. 4. Conclusion and perspectives
Fig. 3. Structural stability of Eco tRNAAsp and Hsa mt-tRNAAsp. Free energies were calculated with the RNAeval program of the Vienna RNA package, with fixed secondary structure cloverleaf constraints and for a temperature of 15 C. Wild-type and transplanted mammalian mitochondrial tRNAAsp variants cloverleaf structures are approximately 2-fold less stable than Eco tRNAAsp (wild-type and variants).
calculated with the RNAeval program of the Vienna RNA package. The calculations do not take into account the D-/T-loop interactions nor any tertiary interaction and are performed on unmodified transcript sequences; they highlight impressive differences between the stability of Eco tRNA derivatives (about 42 kcal/mol) and mt-tRNA variants (about 24 kcal/mol) at 15 C (see free energies at different temperatures in Supplementary Table 1), however, supporting a far higher stability of Eco tRNAs than mttRNAs. These calculations are also in agreement with large differences in melting temperatures measured for in vitro transcribed “classical” tRNAs (about 60 C for yeast tRNAPhe [36]; and yeast tRNAAsp [20]) and Hsa mt-tRNA (e.g. 37 C for tRNALys [37]). A small population of Hsa mt-tRNAAsp also folds into alternative structures such as an incomplete cloverleaf and an extended hairpin [23]. The free energies of these structures are even weaker than that of the cloverleaf fold, with DG ¼ 18 kcal/mol and 13 kcal/mol respectively. We propose that the about two-fold lower stability of the mttRNAs as compared to Eco tRNAAsp represents a strong barrier for proper accommodation to the Eco AspRS. This enzyme is probably unable to deal properly with the too large intrinsic flexibility of the mt-tRNA substrate. A variant was prepared in which base-pair U31eA39 in the anticodon stem was replaced by C31eG39 within the most efficient mt-tRNAAsp derivative constructed so far, namely mt-tRNAAspidec/ DT/U11eA24 (L ¼ 260-fold). Base-pair 31e39 was chosen since a similar substitution, A31$C39 to G31eC39 in Hsa mt-tRNALeu(UUR), was previously shown to convert an otherwise unfolded domain into a helical folding of both anticodon and D-stems [38,39]. The resulting variant mt-tRNAAspidec/DT/U11eA24/C31eG39 is chargeable by both synthetases (data not shown) and indeed, causes a 10-fold increase in the catalytic rate constant of aspartylation by Eco AspRS (Table 2). This positive effect is due to a 35-fold increase in KM so that the final aspartylation efficiency remains 635-fold lower than for wt Eco tRNAAsp. A systematic search for individual or combined base-pairs of Hsa mt-tRNAAspidec/DT/ U11eA24 to be converted from weak AeU or GeU pairs into more stable GeC pairs, would likely make possible the full conversion of the mt-tRNA into an optimal substrate for the Eco AspRS. However, this would require conversion of about 9 pairs to
The case study presented herein pinpoints a number of molecular reasons for the cross-aminoacylation barrier between a mammalian mt-tRNA and a bacterial aaRS. As a result we propose that the absence of aminoacylation of Hsa mt-tRNAAsp by Eco AspRS is linked to three major drawbacks: (i) incompleteness of features defining classical aminoacylation rules (including identity elements and a structural motif), (ii) the presence of at least one idiosyncratic antideterminant or neutral element and possible additional context elements, and (iii) too large intrinsic and global conformational flexibility hindering optimal adaptation to the synthetase. Accordingly, and as opposed to other cross-aminoacylation barriers discussed in the Introduction and dealing with tRNAs from various kingdoms and various species but all of same “classical” structural organization, conversion of a mt-tRNA into an efficient substrate for a bacterial synthetase cannot be solved uniquely by considering general aminoacylation rules but is also based on thermodynamic properties of the tRNA. The present data have been obtained on in vitro transcribed tRNAs, deprived of post-transcriptional modifications. Posttranscriptional modifications are known to restrict the landscape of alternative structures of tRNAs (as demonstrated in the case of Hsa mt-tRNALys [40,41]) and to stabilize RNA conformations (e.g. [20,36]). Native Hsa mt-tRNAAsp has only 4 modifications (m1A9, m2G10, J27, Q34 [23]) and native Eco tRNAAsp has 9 modifications (s4U8, D16, D20, D20:1, Q34, m2A37, m7G46, T54, J55; [42]). Even if not all modifications lead to stabilization (e.g. s4U or D increase local flexibility), it is likely that while both native tRNAs fold into the same structure, they can still be distinguished by differences in stability (i.e., the Eco native tRNAAsp being more stable than the native mt-tRNAAsp) and that the general conclusions derived above likely remain valid. The fact that variants derived from mt-tRNAAsp coming from 4 additional mammals are aminoacylated to different extents further suggests that detailed idiosyncratic structural features also contribute to the cross-aminoacylation barrier with Eco AspRS. Base-pair 11e24 is one of these. It is interesting to note that this base-pair, investigated as the “Hirsch suppressor” when mutated in the Eco tRNATrp, was recently shown to act through an increase in flexibility on the ribosome [43]. The contribution of the tRNA partner to successful induced-fit to the synthetase has been reported previously (e.g. [44]). In the case of mt-tRNAs, however, this is amplified in an unprecedented way. Mt-tRNAs are coded by both strands of the mammalian mtgenome which are of highly different nucleotide composition (Grich or “heavy” strand; C-rich or “light” strand). In combination with the high mutation rate of the mt-DNA, the population of mttRNAs covers a large range of structural diversity, with “light” and “heavy” tRNAs in terms of nucleotide content, and with “classical” to “highly degenerated” tRNAs in regard of 3D structures and stability [22,24]. It can thus be anticipated that the barriers for cross-aminoacylation of the set of “light” tRNAs by bacterial enzymes would display the same general trend as those deciphered for mt-tRNAAsp. Restriction of the set of identity elements within mammalian mt-tRNAs has been observed for other aminoacylation systems investigated so far, namely those for tyrosine [45] and serine [46,47]. Furthermore, all tRNAs considered in the pioneering work of Kumazawa and colleagues (Table 1, [7,8]) are “light” tRNAs that share the structural properties of Hsa tRNAAsp. While the present work highlights peculiar tRNA properties that contribute to restricted recognition by aaRSs, an intriguing unsolved question concerns the properties of the bacterial and
1096
A. Fender et al. / Biochimie 94 (2012) 1090e1097
mitochondrial synthetases responsible for their different behaviours when facing the same tRNA substrate. Initial answers to this question come from genetic approaches which allow conversion of a bacterial synthetase into a yeast mitochondrial enzyme [48,49]. A fine-tuned comparative analysis of both Hsa mt-AspRS and Eco AspRS has already shown how the mt-AspRS deals with the absence of the major aspartate identity element G73, revealing subtle local evolutionary adaptation by a point mutation in the binding pocket [14]. Full understanding requires further explorations and in particular access to crystallographic structures and refined thermodynamic investigations of tRNA/synthetase partnerships. Work is in progress along these lines.
Acknowledgements We thank R. Giegé and L. Levinger for critical reading and helpful comments on the manuscript, G. Eriani, H.D. Becker and D. Kern for gift of material. This work was supported by Centre National de la Recherche Scientifique, Université de Strasbourg, Association Française contre les Myopathies and Action Concertée Incitative (ACI-BCMS 146). A.F. was supported by a fellowship from Ministère de l’Enseignement Supérieur et de la Recherche and F.J. by the German Academic Exchange Service (DAAD D/10/43622).
Appendix. Supplementary material Supplementary data associated with this article can be found, in the online version, at 10.1016/j.biochi.2012.02.030.
References [1] R. Giegé, M. Sissler, C. Florentz, Universal rules and idiosyncratic features in tRNA identity, Nucleic Acids Res. 26 (1998) 5017e5035. [2] P.J. Beuning, K. Musier-Forsyth, Transfer RNA recognition by aminoacyl-tRNA synthetases, Biopolymers 52 (1999) 1e28. [3] R. Giegé, M. Frugier, Transfer RNA structure and identity, in: J. Lapointe, L. Brakier-Gringas (Eds.), Translation Mechanisms, Landes Sciences, Georgetown, TX, 2003, pp. 1e24. [4] R. Giegé, B. Rees, Aspartyl-tRNA synthetases, in: M. Ibba, C. Francklyn, S. Cusack (Eds.), Aminoacyl-tRNA Synthetases, Landes Bioscience, Georgetown, TX, 2005, pp. 210e226. [5] M.E. Saks, J.R. Sampson, Variant minihelix RNAs reveal sequence-specific recognition of the helical tRNASer acceptor stem by E. coli seryl-tRNA synthetase, EMBO J. 15 (1996) 2843e2849. [6] M. Frugier, M. Helm, B. Felden, R. Giegé, C. Florentz, Sequences outside recognition sets are not neutral for tRNA aminoacylation: evidence for nonpermissive combinations of nucleotides in the acceptor stem of yeast tRNAPhe, J. Biol. Chem. 273 (1998) 11605e11610. [7] Y. Kumazawa, T. Yokogawa, E. Hasegawa, K.-I. Miura, K. Watanabe, The aminoacylation of structurally variant phenylalanine tRNAs from mitochondria and various nonmitochondrial sources by bovine mitochondrial phenylalanyltRNA synthetase, J. Biol. Chem. 264 (1989) 13005e13011. [8] Y. Kumazawa, H. Himeno, K.-I. Miura, K. Watanabe, Unilateral aminoacylation specificity between bovine mitochondria and eubacteria, J. Biochem. 109 (1991) 421e427. [9] B. Brindefalk, J. Viklund, D. Larsson, M. Thollesson, S.G. Andersson, Origin and evolution of the mitochondrial aminoacyl-tRNA synthetases, Mol. Biol. Evol. 24 (2007) 743e756. [10] L. Bonnefond, A. Fender, J. Rudinger-Thirion, R. Giegé, C. Florentz, M. Sissler, Towards the full set of human mitochondrial aminoacyl-tRNA synthetases: characterization of AspRS and TyrRS, Biochemistry 44 (2005) 4805e4816. [11] M. Sissler, J. Pütz, F. Fasiolo, C. Florentz, Mitochondrial aminoacyl-tRNA synthetases, in: M. Ibba, C. Francklyn, S. Cusack (Eds.), Aminoacyl-tRNA Synthetases, Landes Biosciences, Georgetown, TX, 2005, pp. 271e284. [12] G. Eriani, M. Delarue, O. Poch, J. Gangloff, D. Moras, Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs, Nature 347 (1990) 203e206. [13] S. Cusack, C. Berthet-Colominas, M. Härtlein, N. Nassar, R. Leberman, A second class of synthetase structure revealed by X-ray analysis of Escherichia coli seryl-tRNA synthetase at 2.5 Å, Nature 347 (1990) 249e255. [14] A. Fender, C. Sauter, M. Messmer, J. Pütz, R. Giegé, C. Florentz, M. Sissler, Loss of a primordial identity element for a mammalian mitochondrial aminoacylation system, J. Biol. Chem. 281 (2006) 15980e15986.
[15] J. Pütz, J.D. Puglisi, C. Florentz, R. Giegé, Identity elements for specific aminoacylation of yeast tRNAAsp by cognate aspartyl-tRNA synthetase, Science 252 (1991) 1696e1699. [16] H.D. Becker, R. Giegé, D. Kern, Identity of prokaryotic and eukaryotic tRNAAsp for aminoacylation by aspartyl-tRNA synthetase from Thermus thermophilus, Biochemistry 35 (1996) 7447e7458. [17] H.D. Becker, J. Reinbolt, R. Kreutzer, R. Giegé, D. Kern, Existence of two distinct aspartyl-tRNA synthetases in Thermus thermophilus. Structural and biochemical properties of the two enzymes, Biochemistry 36 (1997) 8785e8797. [18] T. Hasegawa, H. Himeno, H. Ishikura, M. Shimizu, Discriminator base of tRNAAsp is involved in amino acid acceptor activity, Biochem. Biophys. Res. Commun. 163 (1989) 1534e1538. [19] N. Nameki, K. Tamura, H. Himeno, H. Asahara, T. Hasegawa, M. Shimuzu, E. coli tRNAAsp recognition mechanism differing from that of the yeast system, Biochem. Biophys. Res. Commun. 189 (1992) 856e862. [20] V. Perret, A. Garcia, J.D. Puglisi, H. Grosjean, J.-P. Ebel, C. Florentz, R. Giegé, Conformation in solution of yeast tRNAAsp transcripts deprived of modified nucleotides, Biochimie 72 (1990) 735e744. [21] P. Fechter, J. Rudinger, R. Giegé, A. Théobald-Dietrich, Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity, FEBS Lett. 436 (1998) 99e103. [22] M. Helm, H. Brulé, D. Friede, R. Giegé, J. Pütz, C. Florentz, Search for characteristic structural features of mammalian mitochondrial tRNAs, RNA 6 (2000) 1356e1379. [23] M. Messmer, J. Pütz, T. Suzuki, T. Suzuki, C. Sauter, M. Sissler, C. Florentz, Tertiary network in mammalian mitochondrial tRNAAsp revealed by solution probing and phylogeny, Nucleic Acids Res. 37 (2009) 6881e6895. [24] R. Giegé, F. Jühling, J. Pütz, P. Stadler, C. Sauter, C. Florentz, Structure of transfer RNAs: similarity and variability, Wiley Interdiscip. Rev. RNA 3 (2012) 37e61. [25] M. Frugier, C. Florentz, R. Giegé, Efficient aminoacylation of resected RNA helices by class II aspartyl-tRNA synthetase dependent on a single nucleotide, EMBO J. 13 (1994) 2218e2226. [26] H. Choi, G. Kay, J.A. Schneider, S. Otten, W.H. McClain, Recognition of acceptorstem structure of tRNAAsp by Escherichia coli aspartyl-tRNA synthetase, RNA 9 (2003) 386e393. [27] J. Pütz, B. Dupuis, M. Sissler, C. Florentz, Mamit-tRNA, a database of mammalian mitochondrial tRNA primary and secondary structures, RNA 13 (2007) 1184e1190. [28] G. Dirheimer, G. Keith, P. Dumas, E. Westhof, Primary, secondary and tertiary structures of tRNAs, in: D. Söll, U.L. RajBhandary (Eds.), tRNA: Structure, Biosynthesis, and Function, Am. Soc. Microbiol. Press, Washington, DC, 1995, pp. 93e126. [29] S. Eiler, A.C. Dock-Bregeon, L. Moulinier, J.-C. Thierry, D. Moras, Synthesis of aspartyl-tRNAAsp in Escherichia coli-a snapshot of the second step, EMBO J. 18 (1999) 6532e6541. [30] J. Pütz, J.D. Puglisi, C. Florentz, R. Giegé, Additive, cooperative and anticooperative effects between identity nucleotides of a tRNA, EMBO J. 12 (1993) 2949e2957. [31] S. Hauenstein, C.M. Zhang, Y.M. Hou, J.J. Perona, Shape-selective RNA recognition by cysteinyl-tRNA synthetase, Nat. Struct. Mol. Biol. 11 (2004) 1134e1141. [32] R. Giegé, Toward a more complete view of tRNA biology, Nat. Struct. Mol. Biol. 15 (2008) 1007e1014. [33] A. Sethi, J. Eargle, A.A. Black, Z. Luthey-Schulten, Dynamical networks in tRNA:protein complexes, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 6620e6625. [34] R.W. Alexander, J. Eargle, Z. Luthey-Schulten, Experimental and computational determination of tRNA dynamics, FEBS Lett. 584 (2010) 376e386. [35] B. Rees, G. Webster, M. Delarue, M. Boeglin, D. Moras, Aspartyl-tRNA synthetase from Escherichia coli: flexibility and adaptability to the substrates, J. Mol. Biol. 299 (2000) 1157e1164. [36] J.R. Sampson, O.C. Uhlenbeck, Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 1033e1037. [37] C. Florentz, H. Brulé, M. Helm, R. Giegé, Structural studies of human mitochondrial tRNAs, in: P. Lestienne (Ed.), Mitochondrial Diseases, Springer Verlag, Heidelberg, 1999, pp. 59e72. [38] B. Sohm, M. Sissler, H. Park, M.P. King, C. Florentz, Recognition of human mitochondrial tRNALeu(UUR) by its cognate leucyl-tRNA synthetase, J. Mol. Biol. 339 (2004) 17e29. [39] E.I. Zagryadskaya, S.O. Kelley, Combinatorial analysis of loop nucleotides in human mitochondrial tRNALeu(UUR), Biochemistry 44 (2005) 233e242. [40] M. Helm, H. Brulé, F. Degoul, C. Cepanec, J.-P. Leroux, R. Giegé, C. Florentz, The presence of modified nucleotides is required for cloverleaf folding of a human mitochondrial tRNA, Nucleic Acids Res. 26 (1998) 1636e1643. [41] Y. Motorin, M. Helm, tRNA stabilization by modified nucleotides, Biochemistry 49 (2010) 4934e4944. [42] F. Jühling, M. Mörl, R.K. Hartmann, M. Sprinzl, P.F. Stadler, J. Pütz, tRNAdb 2009: compilation of tRNA sequences and tRNA genes, Nucleic Acids Res. 37 (2009) D159eD162. [43] X. Agirrezabala, E. Schreiner, L.G. Trabuco, J. Lei, R.F. Ortiz-Meoz, K. Schulten, R. Green, J. Frank, Structural insights into cognate versus near-cognate discrimination during decoding, EMBO J. 30 (2011) 1497e1507.
A. Fender et al. / Biochimie 94 (2012) 1090e1097 [44] L. Moulinier, S. Eiler, G. Eriani, J. Gangloff, J.-C. Thierry, K. Gabriel, W.H. McClain, D. Moras, The structure of an AspRS-tRNAAsp complex reveals a tRNA-dependant control mechanism, EMBO J. 20 (2001) 5290e5301. [45] L. Bonnefond, M. Frugier, R. Giegé, J. Rudinger-Thirion, Human mitochondrial TyrRS disobeys the tyrosine idenity rules, RNA 11 (2005) 558e562. [46] T. Ueda, Y. Yotsumoto, K. Ikeda, K. Watanabe, The T-loop region of animal mitochondrial tRNASer(AGY) is a main recognition site for homologous seryltRNA synthetase, Nucleic Acids Res. 20 (1992) 2217e2222.
1097
[47] S. Chimnaronk, M. Gravers Jeppesen, T. Suzuki, J. Nyborg, K. Watanabe, Dualmode recognition of noncanonical tRNASer by seryl-tRNA synthetase in mammalian mitochondria, EMBO J. 24 (2005) 3369e3379. [48] H.Y. Huang, Y. Kuei, H.Y. Chao, S.J. Chen, L.S. Yeh, C.C. Wang, Cross-species and cross-compartmental aminoacylation of isoaccepting tRNAs by a class II tRNA synthetase, J. Biol. Chem. 281 (2006) 31430e31439. [49] W.C. Chiu, C.P. Chang, C.C. Wang, Evolutionary basis of converting a bacterial tRNA synthetase into a yeast cytoplasmic or mitochondrial enzyme, J. Biol. Chem. 284 (2009) 23954e23960.