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Opinion TRENDS in Biochemical Sciences Vol.30 No.12 December 2005

Amino acid specificity in

Taraka Dale and Olke C. Uhlenbeck

Department of Biochemistry, , and Cell Biology, Northwestern University, Evanston, IL 60208, USA

Recent structural and biochemical experiments indicate For example, in the course of deducing the recognition that bacterial Tu and the ribosomal rules of aaRSs, several amber-suppressor tRNA bodies A-site show specificity for both the and the were deliberately mutated such that they were amino- tRNA portions of their aminoacyl-tRNA (aa-tRNA) acylated by a different aaRS, and the resulting ‘identity- substrates. These data are inconsistent with the swapped’ tRNAs were shown to insert the new amino acid traditional view that tRNAs are generic adaptors in into [7,8]. In addition, suppressor tRNAs esterified translation. We hypothesize that each tRNA sequence with O30 different unnatural amino acids have been has co-evolved with its cognate amino acid, such that all successfully incorporated into protein [9]. Together, these aa-tRNAs are translated uniformly. data suggest that the translational apparatus lacks specificity for different amino acids, once they are Introduction esterified onto tRNA. The mechanism of protein synthesis is traditionally In a few isolated cases, however, the translation considered to have two phases with different specificities machinery seems to show specificity for the esterified towards the 20 amino acid side chains (Figure 1). In the amino acid. A prominent example occurs in the transami- first phase, each amino acid is specifically recognized by dation pathway, which is used as an alternative to GlnRS its cognate aminoacyl-tRNA synthetase (aaRS) and to produce Gln-tRNAGln in many and esterified to the appropriate tRNA to form an aminoacyl- [10,11]. In this pathway, tRNAGln is first misacylated by tRNA (aa-tRNA). In the second phase, all of the different GluRS to form Glu-tRNAGln and then reacted with a aa-tRNAs are funneled into the translational machinery specific amidotransferase to produce Gln-tRNAGln. by binding elongation factor Tu$GTP (EF-Tu$GTP; EF-1 Because organisms using this pathway do not show mis- in eukaryotes) to form a ternary complex, which sub- incorporation of glutamic acid at glutamine codons, it sequently binds to the . The traditional view has seems that the misacylated Glu-tRNAGln intermediate of been that the components of this second phase are not this pathway is not translated. However, in vitro exper- specific for the type of amino acid, and that tRNAs are iments show that, although Gln-tRNAGln can bind EF-Tu, generic adaptors that are entirely specified by the anti- Glu-tRNAGln binds poorly [12]. As a result, the amido- codon. As summarized by Woese [1], this view that tRNAs transferase can successfully compete with EF-Tu for the are adaptors which connect the amino acid with the misacylated Glu-tRNAGln. Thus, in this case, the transla- anticodon was hypothesized on theoretical grounds by tional machinery seems to discriminate against certain Crick in 1958 [2] and immediately accepted as the misacylated tRNAs. paradigm. This lack of specificity for the amino acid in the second phase of translation ensures that all amino Hypothesis: amino acid specificity in translation acids are incorporated into protein with similar efficien- promotes tRNA diversity cies and rates, despite their characteristic differences in Although the bacterial translation apparatus shows little size, charge and hydrophobicity. specificity for cognate aa-tRNAs, it does show specificity Experiments showing that misacylated tRNAs can be for the esterified amino acid portion of each aa-tRNA. incorporated into protein supported the paradigm by In the case of EF-Tu, this specificity is only observed by a suggesting that the translational machinery does not quantitative analysis of the binding properties of mis- recognize the esterified amino acid of aa-tRNA. The classic acylated tRNAs. In the case of the ribosome, the X-ray Chapeville experiment using Raney Nickel to convert structure and the majority of the biochemical data suggest Cys-tRNACys to Ala-tRNACys found that alanine was that the ribosomal A-site also shows specificity for binding incorporated at the cysteine codons in an in vitro transla- the esterified amino acid. We hypothesize that, for each tion assay [3]. This result demonstrated that the trans- aa-tRNA to function equivalently in translation, each lational machinery is unable to distinguish an incorrect tRNA sequence has evolved to adjust its affinity for EF-Tu from a correct amino acid. Many additional examples of and for the ribosome in a way that compensates for the incorporation of amino acids from misacylated tRNAs into particular affinity of its cognate amino acid. As a result, protein have since been reported [4–6]. Perhaps the most tRNAs are not generic, interchangeable adaptors, but extensive experiments evaluating the incorporation of have individually evolved to be translated uniformly. misacylated tRNAs relied on measuring the extent of suppression of nonsense codons by suppressor tRNAs. Crystal structures of three amino-acid-binding pockets

Corresponding author: Uhlenbeck, O.C. ([email protected]). Amino acid binding in translation can be pictured using Available online 2 November 2005 several available X-ray crystal structures. Figure 2 www.sciencedirect.com 0968-0004/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2005.10.006 660 Opinion TRENDS in Biochemical Sciences Vol.30 No.12 December 2005

(a) Phase I: aa-tRNA synthesis

aaRS

PPi

Amino acid

ATP

AMP aaRS tRNA

(b) Phase II: translation aa-tRNA

Ternary complex EF-Tu GTP formation EPA

Ribosome with empty A-site Ternary complex binding and decoding

Ribosome with EPA empty A-site

EF-G GDP EPA + P i Translocation GTP Pi EF-G GTP

Peptidyl transfer Accommodation

EPA EPA EPA

EF-Tu GDP

Ti BS

Figure 1. The two phases of translation. (a) Phase I, the specific phase, involves the ATP-dependent aminoacylation of a given tRNA by its cognate aminoacyl-tRNA synthetase (aaRS). An example reaction (red) of the formation of Gln-tRNAGln by GlnRS is shown. Although other tRNAs (other colors) have a similar overall shape, their different nucleotide sequences and repertoire of post-transcriptional modifications preclude them from interacting with GlnRS, and only glutamine precisely fits into the amino-acid- www.sciencedirect.com Opinion TRENDS in Biochemical Sciences Vol.30 No.12 December 2005 661 compares the amino-acid-binding pockets of TyrRS [13], pockets of EF-Tu and the ribosomal A-site could easily EF-Tu [14] and the A-site of 50S [15], each of show specificity for the different amino acid side chains. which was co-crystallized with an amino acid or amino acid analog. In the case of TyrRS, the bound tyrosine The hidden specificities of EF-Tu precisely fits into the pocket and makes numerous As would be expected for a protein that must bind multiple hydrogen bonds and Van der Waals contacts with the substrates, EF-Tu binds to all cognate aa-tRNAs within a protein (Figure 2a). In the case of EF-Tu, which is narrow range of affinities [17,18]. This uniform binding is Phe complexed with Phe-tRNA , the esterified phenyl- consistent with the idea that EF-Tu is a non-specific alanine fits in a large crevice in the protein and seems to binding protein that does not discriminate between stack with His67 (Figure 2b). In addition, several different amino acids or tRNA sequences. However, other hydrogen bonds form between the a-amino group of the experiments using misacylated tRNAs have revealed a esterified phenylalanine and EF-Tu. Finally, the amino- ‘hidden’ specificity of EF-Tu for both the amino acid side acid-binding pocket in the ribosomal A-site differs from chain and the tRNA. For example, early studies showed the previous two in that it is composed entirely of rRNA. In that Phe-tRNALys binds to EF-Tu approximately fivefold this case, the p-methoxyphenyl portion of the tighter than the cognate Lys-tRNALys [19,20]. An expanded binds in a cleft formed by two rRNA bases, presumably study used four tRNAs and four amino acids to form the 16 deriving stability via stacking interactions (Figure 2c). possible combinations of aa-tRNAs [21]. As expected, the At first glance, the three amino-acid-binding pockets four cognate aa-tRNAs bound EF-Tu with similar affi- seem to be well suited for their required functions. TyrRS, nities; however, the binding affinities of the 12 misacylated like all aaRSs, uses its pocket to specifically select its tRNAs to EF-Tu varied dramatically from 13 times weaker cognate amino acid from the 19 non-cognate amino acids, to 400 times stronger than the cognate aa-tRNAs. which is crucial for properly pairing the amino acid and Subsequently, tRNAPhe was misacylated with 13 different tRNA during aa-tRNA formation. Smaller amino acids amino acids, and their affinities for EF-Tu were found to cannot form all of the stabilizing hydrogen bonds, whereas vary from 1.9 nM to 150 nM (w80-fold) [22]. Furthermore, larger amino acids will not fit into the pocket. By contrast, 19 different tRNA bodies misacylated with the same amino EF-Tu and the ribosomal A-site have pockets that are acid (valine) and displayed a 700-fold range in affinities large enough to fit any amino acid side chain, which is (0.44–310 nM) [23]. Taken together, these data demon- consistent with their need to accept all of the different strate that both the amino acid side chain and the tRNA aa-tRNAs as substrates. However, because the pockets of body make highly varied and independent thermodynamic both EF-Tu and the A-site seem to form stacking contributions to EF-Tu binding, and that these contri- interactions with aromatic amino acids, these amino butions compensate for one another such that the overall acids might bind with higher affinity than smaller amino binding of cognate aa-tRNAs is uniform (Figure 3). Thus, acids, which cannot stack. This raises several questions: the apparent lack of specificity of EF-Tu for cognate aa- † How do other amino acid side chains fit into the EF-Tu tRNAs is actually the result of two specific, but opposing, and A-site pockets? interactions between the protein and the aa-tRNA. † Do the residues that form the pockets retain a rigid The experiments involving misacylated tRNAs and structure, or do they rearrange to accommodate the EF-Tu demonstrate that, rather than being a generic different amino acids? adaptor, each tRNA has an important role in ensuring † What are the binding affinities of the different amino uniform binding of aa-tRNA to EF-Tu. Parts of the acids for each pocket? sequence of each tRNA have evolved to compensate for From the limited data, it seems that some repositioning the variable contribution of its esterified amino acid to the of pocket residues can take place. An X-ray crystal overall binding affinity. For example, to compensate for structure of EF-Tu complexed with Cys-tRNACys shows the weak binding of alanine, tRNAAla contains sequence that small rearrangements of the amino-acid-binding elements that ensure that it binds to EF-Tu stronger than pocket occur to enable the sulfhydryl group to pack tRNAGln, which is associated with the strongly binding against Asn285 instead of stacking with His67 [16]. glutamine. Because the co-crystal structure of the ternary However, the consequences of this rearrangement with complex shows that the protein primarily interacts with regard to amino acid specificity are unclear. Thus, the the phosphodiester backbone of the acceptor and T stems crystal structures indicate that the amino-acid-binding of tRNA [14,16], it is likely that sequence differences

binding pocket of GlnRS. For each aaRS, the aminoacylation reaction is highly specific for both the amino acid and the tRNA, such that the incorrect formation of a misacylated tRNA is estimated to be one in 10 000 [11]. (b) In phase II, the non-specific phase of translation, all aa-tRNAs are funneled into a common translational apparatus (grey). Each aa-tRNA forms a ternary complex with elongation factor Tu (EF-Tu) and GTP, and binds to the ribosome in process termed decoding. Correct codon–anticodon pairing between the A-site mRNA codon and the tRNA anticodon during decoding activates the GTPase activity of EF-Tu [43,44], and GTP hydrolysis occurs. A subsequent conformational change is induced in EF-Tu, which results in the release of aa-tRNA and enables the acceptor end of the aa-tRNA to move into the A-site in a process termed accommodation. After accommodation, the growing polypeptide esterified to the P-site-bound tRNA is transferred to the A-site-bound tRNA, elongating the chain by one amino acid. With the aid of elongation factor G (EF-G), the deacylated P-site tRNA is then translocated to the E-site, and the A-site-bound tRNA is translocated to the P-site. The ribosomal A-site is then available for binding to the next ternary complex. These steps were discerned from a series of kinetic measurements [27]. The structures of GlnRS and the GlnRS–tRNAGln complex are from PDB file 1GSG [45]. The tRNA, EF-Tu$GTP, and ternary complex structures are from PDB file 1TTT [14]. EF-Tu$GDP structure is from PDB file 1TUI [46]. The complex structure of the ribosome and detailed positioning of the aa-tRNAs on the ribosome were omitted for simplicity; molecules are not drawn to scale. www.sciencedirect.com 662 Opinion TRENDS in Biochemical Sciences Vol.30 No.12 December 2005

Figure 2. Amino-acid-binding pockets in translation. All three pockets are viewed from the solvent-accessible side. (a) X-ray crystal structure of the tyrosine-binding pocket of TyrRS (PDB code: 1X8X) [13]. The surface representation of the pocket (i) shows a precise fit between the bound tyrosine (green) and the pocket (colored by electrostatic potential: red, negative; blue, positive). The stick representation (ii) reveals putative hydrogen bonds (broken lines) between the p-hydroxyl group of the bound tyrosine and protein residues Tyr37 and Asp182, which aid in achieving substrate specificity. In addition, the a-amino group of the bound tyrosine is within hydrogen- bonding distance of Gln179, Tyr175 and Asp81, which might further stabilize the position of the substrate. (b) X-ray crystal structure of the amino-acid-binding pocket on the surface of Thermus aquaticus EF-Tu (PDB code: 1TTT) [14]. The 30-terminal adenosine and the esterified phenylalanine of Phe-tRNAPhe are shown (green). The surface representation (i) shows the esterified phenylalanine bound in a spacious pocket that seems to be large enough to accommodate all 20 amino acids. The stick representation of the pocket (ii) shows that the a-amino group of the esterified amino acid can form hydrogen bonds with main-chain protein residues Asn285 and His273. These interactions can be formed with all amino acids except proline. (c) X-ray crystal structure of puromycin (green) bound to the A-site of the 50S subunit of Haloarcula marismortui ribosomes (PDB code: 1KQS) [15]. Puromycin is an analogous to the 30-terminal adenosine and esterified amino acid of an aa-tRNA. Surface (i) and stick (ii) representations show that the large, aromatic p-methoxyphenyl side chain is in a large pocket formed on one side by the imperfectly stacked bases A2486 and C2487 (A2451 and C2452 in E. coli). Aromatic amino acids should bind to this pocket better than small aliphatic groups [47]. www.sciencedirect.com Opinion TRENDS in Biochemical Sciences Vol.30 No.12 December 2005 663 between tRNAGln and tRNAAla in this part of the molecule experiments need to be conducted to better establish the are responsible for the differences in affinity. However, the specificity of the ribosome for different amino acids. precise manner in which the sequence of an RNA duplex affects the positioning of its phosphodiester backbone is Reconciling previous experiments not completely understood; therefore, the rules for tRNA- Given that EF-Tu and the ribosome seem to show sequence specificity for EF-Tu remain to be established. specificity for the esterified amino acid and the tRNA body, how can one reconcile the experiments (reviewed Does the ribosome display amino acid specificity? here) that indicate that misacylated tRNAs can function Of the steps in the elongation cycle (Figure 1) that could be normally in translation? One suggestion is that, of the sensitive to the identity of the amino acid, the accommo- many possible misacylated tRNAs, only certain pairs of dation step is the best candidate. This step, which might tRNAs and amino acids are likely to show reduced consist of several sub-steps [24], involves the release of the translational efficiency. Good examples are the successful acceptor end of aa-tRNA from EF-Tu$GDP and its large incorporation of Ala-tRNACys [3] and poor incorporation of scale movement into the center of the Glu-tRNAGln [12]. Although their ribosome specificities A-site [25,26]. The accommodation step is rate limiting for are not known, tRNAAla and tRNACys bind to EF-Tu with peptide-bond formation and at least partially rate limiting similar affinities (Figure 3), indicating that Ala-tRNACys for the entire elongation cycle [27,28]. Because accommo- binds to EF-Tu nearly as well as cognate Ala-tRNAAla, and dation involves the release of the esterified amino acid could be efficiently delivered to the ribosome. Moreover, from its specific site on EF-Tu and its entry into the tRNAGlu and tRNAGln, in addition to glutamic acid and potentially specific peptidyl transferase center, it seems glutamine, have different affinities for EF-Tu (Figure 3), likely that the identity of the esterified amino acid could which leads to the prediction that the misacylated Glu- affect this step. tRNAGln binds to EF-Tu weakly. Thus, the binding Whereas a similar rate of accommodation has been properties of EF-Tu for these two misacylated tRNAs observed for two different cognate aa-tRNAs [27,29], might explain their relative activities in translation. In kinetic measurements of misacylated tRNAs to assess other cases, the relative specificity of the ribosome for amino acid specificity at this step are yet to be performed. amino acids might determine the activity of However, some data suggest that the ribosomal A-site misacylated tRNAs. itself is specific for binding different amino acid side Even if a misacylated tRNA is reduced in its transla- chains. Bhuta and coworkers [30] measured the activity of tional efficiency, it will not necessarily lead to a reduced a series of aminoacylated derivatives of the dinucleotide overall yield of protein unless it is used at many codons. CpA in a peptidyl transferase reaction and found that For example, at an average amino acid incorporation time their Km values depended upon the identity of the amino of 50 ms (20 amino acids per second), a protein of 100 acid attached to the CpA derivatives. In addition, Starck amino acids will be elongated in 5 s. If it is assumed that et al. [31] showed that the inhibition efficiency of various the same protein must use a misacylated tRNA four times puromycin derivatives depended on the amino acid side and that the misacylated tRNA is incorporated 15 times chain of the derivative [31]. Because both of these small slower (750 ms), then the same protein will be elongated in molecule derivatives mimic the 30 end of aa-tRNA and 8 s. Such a difference in the incorporation rate of a single bind at the peptidyl transferase center, these experiments amino acid would be easily detectable by rapid-mixing clearly indicate that the ribosome displays specificity for experiments [27] and lead to the conclusion that the different amino acid side chains. Although the data are misacylated tRNA had a substantially reduced transla- limited, the relative affinities of different side chains have tional efficiency. However, this same difference in rate a different hierarchy than found for EF-Tu (Figure 3), might not impact the steady-state levels of the protein which is consistent with their fitting into a different when analyzed in vivo or in an in vitro translation assay. binding pocket. In other words, global analyses of overall protein Experiments that measure the binding affinities of production tend to overestimate the incorporation effi- intact aa-tRNAs to the ribosomal A-site are more ciency of a misacylated tRNA. ambiguous with respect to amino acid specificity than A similar argument might explain why so many experiments using mimics of the 30 end of aa-tRNA. The misacylated suppressor tRNAs seem to be fully active in dissociation rates of eight different tRNAs from the translation. A suppressor tRNA is considered effective if it ribosomal A-site were identical when the tRNAs can efficiently read a single nonsense codon before the were aminoacylated but were quite different when they translation-termination machinery can terminate the were deacylated [32]. This suggests that amino acids make protein. However, because translation termination is differing contributions to the affinity of each aa-tRNA for slow compared with a single elongation step [27,34,35],a the A-site and implies that the A-site displays amino acid suppressor tRNA with a substantially reduced elongation specificity. By contrast, several misacylated tRNAs step can still effectively suppress termination. Thus, a measured in the same assay displayed dissociation rates misacylated tRNA might be active as a suppressor but surprisingly similar to their cognate tRNAs [33]. This might function too poorly to be an active elongator tRNA. either means that the A-site does not show amino acid specificity or that the specificity is masked by a ribosomal Perspective: an evolving view of tRNA conformational change that must occur before the aa- It has long been known that part of the sequence diversity tRNA is released. Clearly, additional binding and kinetic among tRNAs results from a need to function in translation www.sciencedirect.com 664 Opinion TRENDS in Biochemical Sciences Vol.30 No.12 December 2005

(a)

Ala-tRNAAla Gln-tRNAAla

KD = 6.2nM KD = 0.05nM

Ala-tRNAGln Gln-tRNAGln

KD = 260nM KD = 4.4nM

(b) Amino acids tRNAs

Glu WEAK Glu STRONG Asp Asp Thr Ala Ala Asp Leu Gly Gly Gly Ala Lys Val Cys Val Met Leu Met Lys Met Arg Arg Pro Thr Pro Phe Pro Phe Lys Phe Thr Arg Ile Ile Ser Ser Asn Asn Val Tyr Ile Cys Trp Trp Trp Gln

Gln Gln STRONG Tyr WEAK

Ti BS

Figure 3. Thermodynamic compensation by EF-Tu. (a) Experimental affinities of two tRNA bodies esterified with two amino acids [21]. Because the thermodynamic contributions of the amino acid and the tRNA balance one another, Ala-tRNAAla and Gln-tRNAGln bind to EF-Tu with similar affinities. ‘Weakly’ binding alanine is esterified to a cognate ‘strongly’ binding tRNAAla, and ‘strongly’ binding glutamine is esterified to a cognate ‘weakly’ binding tRNAGln. Consequently, misacylated Ala-tRNAGln binds to EF- Tu weakly and Gln-tRNAAla binds to EF-Tu strongly, compared with the cognate aa-tRNAs. (b) Thermodynamic contributions of the different amino acids to EF-Tu$aa-tRNA binding. Nineteen amino acid side chains (data for histidine was not obtained) display a large range of affinities for EF-Tu. The order of thermodynamic contributions for amino acid binding to EF-Tu has been determined experimentally for 13 amino acids (bold) [22], and predicted for 19 amino acids (italics) [48]. tRNAs also display a highly variable range of affinities for EF-Tu [23]. As would be expected from the thermodynamic compensation model, the amino acid and tRNA hierarchies are approximately inversely proportional, but the correlation is not perfect because cognate aa-tRNAs do not bind to EF-Tu identically [17,18]. in a manner that is specific for each codon–anticodon pair. permit uniform tRNA function in translation [42]. In other Residues in the anticodon stem-loop correlate with the words, part of the sequence and modification diversity of identity of the anticodon [36], and mutations of those tRNAs is to compensate for the structural, thermodynamic residues affect the translatability of tRNA [37,38].In and kinetic differences that arise from the need for different addition, many of the diverse post-transcriptional modifi- codon–anticodon pairs to be accommodated in translation. cations in tRNA subtly affect translational efficiency when Here, we have discussed data which suggest that an they are deleted [39–41]. Recently, we suggested that this additional evolutionary source of tRNA sequence and idiosyncratic evolutionary ‘tuning’ of tRNAs is required to modification diversity is the identity of the esterified www.sciencedirect.com Opinion TRENDS in Biochemical Sciences Vol.30 No.12 December 2005 665 amino acid. As a result of the environment of the amino- 21 LaRiviere, F.J. et al. (2001) Uniform binding of aminoacyl-tRNAs to acid-binding pockets of EF-Tu and the ribosome, each elongation factor Tu by thermodynamic compensation. Science 294, amino acid has distinct binding properties. As a conse- 165–168 22 Dale, T. et al. (2004) The affinity of elongation factor Tu for an quence of the need for uniformity, the sequences of the aminoacyl-tRNA is modulated by the esterified amino acid. Biochem- tRNA bodies have evolved to compensate for the amino istry 43, 6159–6166 acid specificity of the translational machinery. Thus, 23 Asahara, H. and Uhlenbeck, O.C. (2002) The tRNA Specificity of tRNAs use diversity to function uniformly. Thermus thermophilus EF-Tu. Proc. Natl. Acad. Sci. U. S. A. 99, 3499–3504 In summary, mounting evidence [1,29,36] demands 24 Blanchard, S.C. et al. (2004) tRNA selection and kinetic proofreading that the traditional view of tRNAs as interchangeable, in translation. Nat. Struct. Mol. Biol. 11, 1008–1014 ‘passive’ adaptors be discarded. 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