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Amino Acid Specificity in Translation

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Amino Acid Specificity in Translation Opinion TRENDS in Biochemical Sciences 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 For example, in the course of deducing the recognition that bacterial elongation factor Tu and the ribosomal rules of aaRSs, several amber-suppressor tRNA bodies A-site show specificity for both the amino acid 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 protein [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 bacteria and archaea 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 ribosome. 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 hydrolysis 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 ribosomes [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 puromycin 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).
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