Amino acid specificity in translation

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Vol.30 No.12 December 2005

Amino acid specificity in translation Taraka Dale and Olke C. Uhlenbeck Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA

Recent structural and biochemical experiments indicate that bacterial elongation factor Tu and the ribosomal A-site show specificity for both the amino acid and the tRNA portions of their aminoacyl-tRNA (aa-tRNA) substrates. These data are inconsistent with the traditional view that tRNAs are generic adaptors in translation. We hypothesize that each tRNA sequence has co-evolved with its cognate amino acid, such that all aa-tRNAs are translated uniformly. Introduction The mechanism of protein synthesis is traditionally considered to have two phases with different specificities towards the 20 amino acid side chains (Figure 1). In the first phase, each amino acid is specifically recognized by its cognate aminoacyl-tRNA synthetase (aaRS) and esterified to the appropriate tRNA to form an aminoacyltRNA (aa-tRNA). In the second phase, all of the different aa-tRNAs are funneled into the translational machinery by binding elongation factor Tu$GTP (EF-Tu$GTP; EF-1 in eukaryotes) to form a ternary complex, which subsequently binds to the ribosome. The traditional view has been that the components of this second phase are not specific for the type of amino acid, and that tRNAs are generic adaptors that are entirely specified by the anticodon. As summarized by Woese [1], this view that tRNAs are adaptors which connect the amino acid with the anticodon was hypothesized on theoretical grounds by Crick in 1958 [2] and immediately accepted as the paradigm. This lack of specificity for the amino acid in the second phase of translation ensures that all amino acids are incorporated into protein with similar efficiencies and rates, despite their characteristic differences in size, charge and hydrophobicity. Experiments showing that misacylated tRNAs can be incorporated into protein supported the paradigm by suggesting that the translational machinery does not recognize the esterified amino acid of aa-tRNA. The classic Chapeville experiment using Raney Nickel to convert Cys-tRNACys to Ala-tRNACys found that alanine was incorporated at the cysteine codons in an in vitro translation assay [3]. This result demonstrated that the translational machinery is unable to distinguish an incorrect from a correct amino acid. Many additional examples of incorporation of amino acids from misacylated tRNAs into protein have since been reported [4–6]. Perhaps the most extensive experiments evaluating the incorporation of misacylated tRNAs relied on measuring the extent of suppression of nonsense codons by suppressor tRNAs. Corresponding author: Uhlenbeck, O.C. ([email protected]). Available online 2 November 2005

For example, in the course of deducing the recognition rules of aaRSs, several amber-suppressor tRNA bodies were deliberately mutated such that they were aminoacylated by a different aaRS, and the resulting ‘identityswapped’ tRNAs were shown to insert the new amino acid into protein [7,8]. In addition, suppressor tRNAs esterified with O30 different unnatural amino acids have been successfully incorporated into protein [9]. Together, these data suggest that the translational apparatus lacks specificity for different amino acids, once they are esterified onto tRNA. In a few isolated cases, however, the translation machinery seems to show specificity for the esterified amino acid. A prominent example occurs in the transamidation pathway, which is used as an alternative to GlnRS to produce Gln-tRNAGln in many bacteria and archaea [10,11]. In this pathway, tRNAGln is first misacylated by GluRS to form Glu-tRNAGln and then reacted with a specific amidotransferase to produce Gln-tRNAGln. Because organisms using this pathway do not show misincorporation of glutamic acid at glutamine codons, it seems that the misacylated Glu-tRNAGln intermediate of this pathway is not translated. However, in vitro experiments show that, although Gln-tRNAGln can bind EF-Tu, Glu-tRNAGln binds poorly [12]. As a result, the amidotransferase can successfully compete with EF-Tu for the misacylated Glu-tRNAGln. Thus, in this case, the translational machinery seems to discriminate against certain misacylated tRNAs. Hypothesis: amino acid specificity in translation promotes tRNA diversity Although the bacterial translation apparatus shows little specificity for cognate aa-tRNAs, it does show specificity for the esterified amino acid portion of each aa-tRNA. In the case of EF-Tu, this specificity is only observed by a quantitative analysis of the binding properties of misacylated tRNAs. In the case of the ribosome, the X-ray structure and the majority of the biochemical data suggest that the ribosomal A-site also shows specificity for binding the esterified amino acid. We hypothesize that, for each aa-tRNA to function equivalently in translation, each tRNA sequence has evolved to adjust its affinity for EF-Tu and for the ribosome in a way that compensates for the particular affinity of its cognate amino acid. As a result, tRNAs are not generic, interchangeable adaptors, but have individually evolved to be translated uniformly. Crystal structures of three amino-acid-binding pockets Amino acid binding in translation can be pictured using several available X-ray crystal structures. Figure 2

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(a) Phase I: aa-tRNA synthesis

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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-acidwww.sciencedirect.com

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compares the amino-acid-binding pockets of TyrRS [13], EF-Tu [14] and the A-site of 50S ribosomes [15], each of which was co-crystallized with an amino acid or amino acid analog. In the case of TyrRS, the bound tyrosine precisely fits into the pocket and makes numerous hydrogen bonds and Van der Waals contacts with the protein (Figure 2a). In the case of EF-Tu, which is complexed with Phe-tRNAPhe, the esterified phenylalanine fits in a large crevice in the protein and seems to stack with His67 (Figure 2b). In addition, several hydrogen bonds form between the a-amino group of the esterified phenylalanine and EF-Tu. Finally, the aminoacid-binding pocket in the ribosomal A-site differs from the previous two in that it is composed entirely of rRNA. In this case, the p-methoxyphenyl portion of the puromycin binds in a cleft formed by two rRNA bases, presumably deriving stability via stacking interactions (Figure 2c). At first glance, the three amino-acid-binding pockets seem to be well suited for their required functions. TyrRS, like all aaRSs, uses its pocket to specifically select its cognate amino acid from the 19 non-cognate amino acids, which is crucial for properly pairing the amino acid and tRNA during aa-tRNA formation. Smaller amino acids cannot form all of the stabilizing hydrogen bonds, whereas larger amino acids will not fit into the pocket. By contrast, EF-Tu and the ribosomal A-site have pockets that are large enough to fit any amino acid side chain, which is consistent with their need to accept all of the different aa-tRNAs as substrates. However, because the pockets of both EF-Tu and the A-site seem to form stacking interactions with aromatic amino acids, these amino acids might bind with higher affinity than smaller amino acids, which cannot stack. This raises several questions: † How do other amino acid side chains fit into the EF-Tu and A-site pockets? † Do the residues that form the pockets retain a rigid structure, or do they rearrange to accommodate the different amino acids? † What are the binding affinities of the different amino acids for each pocket? From the limited data, it seems that some repositioning of pocket residues can take place. An X-ray crystal structure of EF-Tu complexed with Cys-tRNACys shows that small rearrangements of the amino-acid-binding pocket occur to enable the sulfhydryl group to pack against Asn285 instead of stacking with His67 [16]. However, the consequences of this rearrangement with regard to amino acid specificity are unclear. Thus, the crystal structures indicate that the amino-acid-binding

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pockets of EF-Tu and the ribosomal A-site could easily show specificity for the different amino acid side chains. The hidden specificities of EF-Tu As would be expected for a protein that must bind multiple substrates, EF-Tu binds to all cognate aa-tRNAs within a narrow range of affinities [17,18]. This uniform binding is consistent with the idea that EF-Tu is a non-specific binding protein that does not discriminate between different amino acids or tRNA sequences. However, other experiments using misacylated tRNAs have revealed a ‘hidden’ specificity of EF-Tu for both the amino acid side chain and the tRNA. For example, early studies showed that Phe-tRNALys binds to EF-Tu approximately fivefold tighter than the cognate Lys-tRNALys [19,20]. An expanded study used four tRNAs and four amino acids to form the 16 possible combinations of aa-tRNAs [21]. As expected, the four cognate aa-tRNAs bound EF-Tu with similar affinities; however, the binding affinities of the 12 misacylated tRNAs to EF-Tu varied dramatically from 13 times weaker to 400 times stronger than the cognate aa-tRNAs. Subsequently, tRNAPhe was misacylated with 13 different amino acids, and their affinities for EF-Tu were found to vary from 1.9 nM to 150 nM (w80-fold) [22]. Furthermore, 19 different tRNA bodies misacylated with the same amino acid (valine) and displayed a 700-fold range in affinities (0.44–310 nM) [23]. Taken together, these data demonstrate that both the amino acid side chain and the tRNA body make highly varied and independent thermodynamic contributions to EF-Tu binding, and that these contributions compensate for one another such that the overall binding of cognate aa-tRNAs is uniform (Figure 3). Thus, the apparent lack of specificity of EF-Tu for cognate aatRNAs is actually the result of two specific, but opposing, interactions between the protein and the aa-tRNA. The experiments involving misacylated tRNAs and EF-Tu demonstrate that, rather than being a generic adaptor, each tRNA has an important role in ensuring uniform binding of aa-tRNA to EF-Tu. Parts of the sequence of each tRNA have evolved to compensate for the variable contribution of its esterified amino acid to the overall binding affinity. For example, to compensate for the weak binding of alanine, tRNAAla contains sequence elements that ensure that it binds to EF-Tu stronger than tRNAGln, which is associated with the strongly binding glutamine. Because the co-crystal structure of the ternary complex shows that the protein primarily interacts with the phosphodiester backbone of the acceptor and T stems 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 peptide 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

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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 Escherichia coli 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 hydrogenbonding 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 3 0 -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 antibiotic analogous to the 3 0 -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

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between tRNAGln and tRNAAla in this part of the molecule are responsible for the differences in affinity. However, the precise manner in which the sequence of an RNA duplex affects the positioning of its phosphodiester backbone is not completely understood; therefore, the rules for tRNAsequence specificity for EF-Tu remain to be established. Does the ribosome display amino acid specificity? Of the steps in the elongation cycle (Figure 1) that could be sensitive to the identity of the amino acid, the accommodation step is the best candidate. This step, which might consist of several sub-steps [24], involves the release of the acceptor end of aa-tRNA from EF-Tu$GDP and its large scale movement into the peptidyl transferase center of the A-site [25,26]. The accommodation step is rate limiting for peptide-bond formation and at least partially rate limiting for the entire elongation cycle [27,28]. Because accommodation involves the release of the esterified amino acid from its specific site on EF-Tu and its entry into the potentially specific peptidyl transferase center, it seems likely that the identity of the esterified amino acid could affect this step. Whereas a similar rate of accommodation has been observed for two different cognate aa-tRNAs [27,29], kinetic measurements of misacylated tRNAs to assess amino acid specificity at this step are yet to be performed. However, some data suggest that the ribosomal A-site itself is specific for binding different amino acid side chains. Bhuta and coworkers [30] measured the activity of a series of aminoacylated derivatives of the dinucleotide CpA in a peptidyl transferase reaction and found that their Km values depended upon the identity of the amino acid attached to the CpA derivatives. In addition, Starck et al. [31] showed that the inhibition efficiency of various puromycin derivatives depended on the amino acid side chain of the derivative [31]. Because both of these small molecule derivatives mimic the 3 0 end of aa-tRNA and bind at the peptidyl transferase center, these experiments clearly indicate that the ribosome displays specificity for different amino acid side chains. Although the data are limited, the relative affinities of different side chains have a different hierarchy than found for EF-Tu (Figure 3), which is consistent with their fitting into a different binding pocket. Experiments that measure the binding affinities of intact aa-tRNAs to the ribosomal A-site are more ambiguous with respect to amino acid specificity than experiments using mimics of the 3 0 end of aa-tRNA. The dissociation rates of eight different tRNAs from the ribosomal A-site were identical when the tRNAs were aminoacylated but were quite different when they were deacylated [32]. This suggests that amino acids make differing contributions to the affinity of each aa-tRNA for the A-site and implies that the A-site displays amino acid specificity. By contrast, several misacylated tRNAs measured in the same assay displayed dissociation rates surprisingly similar to their cognate tRNAs [33]. This either means that the A-site does not show amino acid specificity or that the specificity is masked by a ribosomal conformational change that must occur before the aatRNA is released. Clearly, additional binding and kinetic www.sciencedirect.com

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experiments need to be conducted to better establish the specificity of the ribosome for different amino acids. Reconciling previous experiments Given that EF-Tu and the ribosome seem to show specificity for the esterified amino acid and the tRNA body, how can one reconcile the experiments (reviewed here) that indicate that misacylated tRNAs can function normally in translation? One suggestion is that, of the many possible misacylated tRNAs, only certain pairs of tRNAs and amino acids are likely to show reduced translational efficiency. Good examples are the successful incorporation of Ala-tRNACys [3] and poor incorporation of Glu-tRNAGln [12]. Although their ribosome specificities are not known, tRNAAla and tRNACys bind to EF-Tu with similar affinities (Figure 3), indicating that Ala-tRNACys binds to EF-Tu nearly as well as cognate Ala-tRNAAla, and could be efficiently delivered to the ribosome. Moreover, tRNAGlu and tRNAGln, in addition to glutamic acid and glutamine, have different affinities for EF-Tu (Figure 3), which leads to the prediction that the misacylated GlutRNAGln binds to EF-Tu weakly. Thus, the binding properties of EF-Tu for these two misacylated tRNAs might explain their relative activities in translation. In other cases, the relative specificity of the ribosome for amino acids might determine the activity of misacylated tRNAs. Even if a misacylated tRNA is reduced in its translational efficiency, it will not necessarily lead to a reduced overall yield of protein unless it is used at many codons. For example, at an average amino acid incorporation time of 50 ms (20 amino acids per second), a protein of 100 amino acids will be elongated in 5 s. If it is assumed that the same protein must use a misacylated tRNA four times and that the misacylated tRNA is incorporated 15 times slower (750 ms), then the same protein will be elongated in 8 s. Such a difference in the incorporation rate of a single amino acid would be easily detectable by rapid-mixing experiments [27] and lead to the conclusion that the misacylated tRNA had a substantially reduced translational efficiency. However, this same difference in rate might not impact the steady-state levels of the protein when analyzed in vivo or in an in vitro translation assay. In other words, global analyses of overall protein production tend to overestimate the incorporation efficiency of a misacylated tRNA. A similar argument might explain why so many misacylated suppressor tRNAs seem to be fully active in translation. A suppressor tRNA is considered effective if it can efficiently read a single nonsense codon before the translation-termination machinery can terminate the protein. However, because translation termination is slow compared with a single elongation step [27,34,35], a suppressor tRNA with a substantially reduced elongation step can still effectively suppress termination. Thus, a misacylated tRNA might be active as a suppressor but might function too poorly to be an active elongator tRNA. Perspective: an evolving view of tRNA It has long been known that part of the sequence diversity among tRNAs results from a need to function in translation

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(a)

Ala-tRNAAla KD = 6.2nM

Gln-tRNAAla KD = 0.05nM

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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 EFTu 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. Residues in the anticodon stem-loop correlate with the identity of the anticodon [36], and mutations of those residues affect the translatability of tRNA [37,38]. In addition, many of the diverse post-transcriptional modifications in tRNA subtly affect translational efficiency when they are deleted [39–41]. Recently, we suggested that this idiosyncratic evolutionary ‘tuning’ of tRNAs is required to www.sciencedirect.com

permit uniform tRNA function in translation [42]. In other words, part of the sequence and modification diversity of tRNAs is to compensate for the structural, thermodynamic and kinetic differences that arise from the need for different codon–anticodon pairs to be accommodated in translation. Here, we have discussed data which suggest that an additional evolutionary source of tRNA sequence and modification diversity is the identity of the esterified

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amino acid. As a result of the environment of the aminoacid-binding pockets of EF-Tu and the ribosome, each amino acid has distinct binding properties. As a consequence of the need for uniformity, the sequences of the tRNA bodies have evolved to compensate for the amino acid specificity of the translational machinery. Thus, tRNAs use diversity to function uniformly. In summary, mounting evidence [1,29,36] demands that the traditional view of tRNAs as interchangeable, ‘passive’ adaptors be discarded. Instead, one must think of tRNAs as active participants in translation that have individually evolved to meet the idiosyncratic needs dictated by their amino acid and anticodon. Acknowledgements This work was supported by National Institutes of Health Grant GM37552 to O.C.U.

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