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Evolving new genetic codes
Opinion
TRENDS in Ecology and Evolution
Vol.19 No.2 February 2004
Evolving new genetic codes Jamie M. Bacher1, Randall A. Hughes2, J. Tze-Fei Wong3 and Andrew D. Ellington1,2 1
Institute of Cellular and Molecular Biology, University of Texas at Austin, 1 University Station A4800, Austin, TX, 78712, USA Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A5300, Austin, TX, 78712, USA 3 Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong 2
Although the genetic code is almost universal, natural variations exist that have caused evolutionary biologists to speculate about codon evolution. There are two predominant hypotheses that specify either a gradual (ambiguous intermediate) or stochastic (codon capture) change in the code. These hypotheses are similar to two biotechnology techniques that have been used to engineer the genetic code: a ‘top down’ approach, in which the whole organism is evolved for the ability to incorporate unnatural amino acids, and a ‘bottom up’ approach, in which aminoacyl-tRNA synthetases and their cognate tRNAs are engineered. The biotechnology experiments provide insights into natural codon evolution, and a combination of these approaches should enable the evolution of organisms that can incorporate unnatural amino acids throughout their proteomes. Although the genetic code was discovered some 40-odd years ago, there are still numerous questions as to how it arose and evolved. One of the first and most famous hypotheses regarding the origin of the code was that it was a ‘frozen accident’ [1], in which associations between codons and amino acids were fixed in place and could change only with difficulty. Since Francis Crick proposed this theory, there have been several alternate explanations for the origins of the code, including stereochemical hypotheses that posit direct interactions between codons and COGNATE AMINO ACIDS (see Glossary) (see, for example [2]) and biosynthetic hypotheses that propose that the code was assigned in parallel to the evolution of amino-acid biosyntheses [3,4]. However, most inquiries have tended to focus on the establishment of the code, rather than on its alteration or maintenance. Although by and large, the code does remain ‘frozen,’ there are several examples of altered genetic codes in nature. One well characterized example is in mitochondrial genomes, where as many as 27 different codon reassignments have been observed [5]. The code can even be changed in free-living organisms; for example, in Candida albicans the ‘universal’ leucine codon CUG is decoded as serine rather than leucine [6]. In addition, novel amino acids, such as selenocysteine [7,8] and pyrrolysine [9], are sometimes incorporated at positions normally encoded by STOP CODONS . There are two principle hypotheses for how natural changes in the code might have evolved: the ‘ambiguous
intermediate’ and ‘codon capture’ hypotheses (reviewed in [10]). In the ‘ambiguous intermediate’ hypothesis [11 – 13], a duplicated and mutated tRNA recognizes a normally NON-COGNATE CODON and inserts its amino acid in competition with the cognate amino acid. Propagation of organisms with ambiguous proteomes could occur if the NON-COGNATE AMINO ACID were either close to selectively neutral or provided a net selective advantage that overcame any deficits in the function of individual proteins. The further evolution of those proteins whose functions were compromised by amino-acid substitutions would eventually repair any minor decreases in fitness. Following the adaptation of individual proteins, a discrete but altered genetic code could be re-established. By contrast, the ‘codon capture’ or ‘disappearing intermediate’ hypothesis [14– 16] posits that certain codons were eliminated by genetic drift throughout genomes that evolved skewed GC or AT contents. Following codon loss, relevant tRNA adaptors became functionless and were deleted. At some later point in evolution, sequence composition changed again, and a different tRNA Glossary Aminoacyl-tRNA synthetase (aaRS): the general name for the group of enzymes responsible for forming the covalent linkage between a specific amino acid and a specific tRNA molecule. Canonical amino acid: an amino acid that is part of the usual complement of 20 amino acids encoded by the genetic code. Charged tRNA: a tRNA that has been covalently linked to an amino acid. Cognate amino acid: an amino acid that is the natural substrate for a particular aminoacyl-tRNA synthetase and corresponds to specific codon(s) in the genetic code. For example, tryptophan is the cognate amino acid of tryptophanyl-tRNA synthetase and has one codon, UGG. Cognate codon: one of 61 nucleotide triplets that encodes for a specific amino acid according to the standard genetic code. For example, UGG is the cognate codon for tryptophan. Directed evolution: a process by which molecules or organisms are subjected to successive cycles of mutation and selection result ingin some desired improved functionality; also known as evolutionary engineering. Non-cognate amino acid: an amino acid that is not the natural substrate for a particular aminoacyl-tRNA synthetase and therefore does not naturally correspond to the codons associated with that amino acid. For example, serine is a non-cognate amino acid of leucyl-tRNA synthetase and, therefore, does not naturally correspond to any of the six codons of leucine. Non-cognate codon: a codon that is not normally assigned to a particular amino acid. For example, the leucine codon CUG is a non-cognate codon to serine. Refractivity: the ability to accept changes; flexibility. Sense codon: a codon that encodes a wild-type amino acid within a gene. Stop codon: a codon that results in termination of an expanding protein. Suppressor tRNA: a tRNA that has been genetically modified to recognize a stop codon; these will function to insert an amino acid at positions that would normally cause an end in protein synthesis. Unnatural amino acid: an amino acid that is not normally encoded by the genetic code; also referred to as an amino-acid analogue.
Corresponding author: Andrew D. Ellington ([email protected]). www.sciencedirect.com 0169-5347/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tree.2003.11.007
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adaptor duplicated, mutated at the anticodon position, and recaptured the codon that had previously disappeared. A variant of this hypothesis suggests that evolutionary pressure on several genotypic characteristics, including genome size and organization as well as composition, influenced codon reassignment [17]. Until recently, very little experimental evidence has been available regarding how mechanistically difficult it might be to reassign amino acids, what the consequences to organismal fitness of such reassignments might be, and how answers to these questions might bear on hypotheses regarding the evolution of the code. Nonetheless, experimental evolution is precisely the tool that should be used to probe these issues, and it has long been used to test theories of evolutionary biology. The combination of molecular biology and experimental evolution was initiated by Spiegelman and colleagues, who developed techniques to amplify the Qb genome entirely in vitro, and then demonstrated how selection pressures, such as faster replication or the introduction of ethidium bromide, could result in the fixation of mutations [18,19]. Furthermore, microorganisms have been evolved over large numbers of generations and in large populations to optimize growth rate [20,21], or to study the evolution of sex [22,23]. With the appropriate restriction of selection pressure, organismal evolution can also be used to evaluate the evolution of single enzymes, such as antibiotic resistance elements [24], or of metabolic pathways [25,26]. As such, microorganisms are effective model systems for the experimental evolution of a broad range of characteristics. Recently, several biotechnology experiments have begun to address these questions. The incorporation of UNNATURAL AMINO ACIDS into organismal proteomes has been achieved by appropriate genetic engineering and DIRECTED EVOLUTION of enzymes specifically involved in the translational apparatus, such as AMINOACYL-TRNA SYNTHETASES (aaRSs). Two different approaches have been used that, for simplicity, can be termed ‘top down’ and ‘bottom up.’ The ‘top down’ approach corresponds roughly to the ‘ambiguous intermediate’ hypothesis, whereas the ‘bottom up’ approach is more consistent with the ‘codon capture’ hypothesis (Box 1). Evolutionary engineering of the genetic code by ‘top down’ approaches Simple selection techniques were sufficient to induce a tryptophan auxotroph of Bacillus subtilis to grow when supplied with the amino acid 4-fluorotryptophan (4fW) in place of the CANONICAL AMINO ACID tryptophan (W; Figure 1) [27]. The chemical substitution of fluorine for hydrogen at the 4 position of W does not drastically alter the size or shape of the amino acid, although its polarity is changed substantially. Moreover, W is the most infrequently utilized amino acid, typically comprising , 1% of an organismal proteome. Even so, the genomic sequence of B. subtilis predicts that there are , 12 600 tryptophans spread over 4100 proteins [28]. It is likely that the functions of at least some of these proteins were critically affected, because studies with proteins that were individually substituted with fluorotryptophan have shown that enzymatic activities could be depressed by as much as an order of magnitude [29,30]. www.sciencedirect.com
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Box 1. The big picture † For the most part, the genetic code has remained ‘frozen’ or universally conserved throughout all taxa; however, a few naturally occurring variations have led to speculation about codon evolution. † There are two principle hypotheses for how natural changes in the code might have evolved: the ‘ambiguous intermediate’ hypothesis and the ‘codon capture’ hypothesis. † Experimental evolution can provide insights into how amino acids can be reassigned and what the effects are of such reassignments on organismal fitness. These experiments also provide additional insight into what biochemical parameters might affect codon evolution. † The ability to alter the genetic code is, in itself, a remarkable feat. The expansion of the chemical functionalities available to an organism should open new options and avenues for evolution, which will enable novel proteins and pathways to be constructed, as well as to expand greatly our understanding of evolutionary processes.
The parental strain of B. subtilis was plated on 4fW and colonies that were uniformly larger were picked. These variants were then mutagenized, and colonies that grew well in the presence of fluorotryptophan but poorly in the presence of tryptophan were picked. After four rounds of coupled positive and negative screening, clone HR15 was found that preferred 4fW for growth, by a ratio of 28:1 based on radioisotope incorporation; this represents a . 2 £ 104 switch in preference between the naturally occurring amino acid and the analogue. Amino-acid analysis revealed that HR15 completely incorporated the analogue into its proteome [27]. The REFRACTIVITY of the genetic code throughout its evolutionary history appears to be wildly at odds with the ease with which it can be manipulated experimentally. Although the initial strain showed limited growth in the presence of the unnatural amino acid, the apparent simplicity of this selection suggests that 4fW had a relatively minor impact on the function of the B. subtilis proteome. Based on these results, it appears that an organism with an ambiguous code can survive and perhaps even prosper if the noncognate amino acid does not greatly impair the function of crucial or numerous proteins. This might imply that ambiguous codon reassignments will be limited, in general, to either structurally similar amino acids or to codons that are used infrequently.
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Figure 1. Structures of tryptophan (a) and 4-fluorotryptophan (b). Tryptophan is shown with the 4, 5, and 6 positions indicated.
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To the extent that organismal fitness is impaired by codon ambiguity, there might be substantial resistance to altering the genetic code, as was found when a set of experiments designed to replace tryptophan with 4fW was performed with Escherichia coli [31]. In these experiments, a tryptophan auxotroph was grown in liquid media in the presence of increasingly higher ratios of 4fW:W, until only 4fW was added at the conclusion of the directed evolution experiment. Several mutations were identified, including changes in tryptophanyl-tRNA synthetase (trpS), the aromatic amino acid permease (aroP) and a repressor of AroP synthesis. However, in contrast to HR15, the evolved E. coli strain did not prefer 4fW for growth, and actually grew extremely poorly on 4fW alone. Although only 4fW was present in the evolved proteome at the limit of the detection methods used (,1% W would have been observed), it appeared that the evolved E. coli still required W for growth. The evolved E. coli had apparently developed the ability to scavenge the very low amounts of W that were present in the medium (commercial 4fW is contaminated with 0.03% W). The level of contaminating W might have been enough to enable the occasional production of proteins containing essential W residues and to therefore support only exceptionally slow growth. Evolutionary paths to amino-acid substitution The relatively small changes in amino-acid preference that were seen with E. coli were surprising, given the earlier results obtained using B. subtilis. The reason for this difference might be related to the number of proteins that would have been critically affected in each organism by the incorporation of the unnatural amino acid. Several lines of evidence indicate that the number might have been relatively small in the experiments with B. subtilis. First, the selection was carried out using a defined but exceptionally rich media that contained nucleotides, vitamins, cofactors and all of the canonical amino acids except W (as well as 4fW). Therefore, to the extent that 4fW incorporation would have compromised enzymes in many biosynthetic pathways, these deficiencies would have gone largely unnoticed. Second, HR15 was derived in only four rounds of colony screening coupled with a relatively low mutation rate and small population size, suggesting that relatively few W residues were critically impaired by replacement with 4fW. Thus, for most of the B. subtilis organismal proteome, the presence of W or 4fW was a largely neutral trait. Furthermore, the ancestral B. subtilis were capable of forming small colonies on the amino-acid analogue. As such, a new ability did not need to be evolved; instead, the selective regime was a higher growth rate under specific conditions. Finally, a selection against growth on W was performed on the B. subtilis variants, further encouraging growth on 4fW by limiting evolutionary options. This ‘negative selection’ might have been extremely important in the successful selection of HR15. By contrast, E. coli was evolved in a minimal media supplemented with 4fW and, thus, many more biosynthetic proteins would have been under selective pressure. In addition, the use of a chemical mutagen during the derivation of HR15 might have provided access to more www.sciencedirect.com
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diverse mutations than were available to the E. coli population. The one relative evolutionary advantage during the E. coli selection was a larger population size (, 107 E. coli serial dilution21 versus . 10 000 B. subtilis plated). The differences between the evolutionary paths taken by the two strains can also be attributed to other factors. The E. coli were evolved in liquid media rather than on plates. It would have therefore been more difficult for individual advantageous mutants to establish themselves because of competition among individuals compared with the direct selection by spatial segregation on plates to which B. subtilis was subjected. Nonetheless, some of the mutations that arose in the evolving E. coli lineage ‘swept’ through a given population within one cycle of serial dilution and growth. In addition, because diffusion was much greater in liquid than on solid media, scavenging strategies would have been more favored in the liquid environment. Finally, no negative selection against growth on W was applied to the evolved E. coli and, thus, robust growth would always have been the most probable option whenever sufficient W was present. Overall, the number of crucial mutations that were required during the E. coli selection to alter amino-acid preference was probably larger than the number that would have been required to generate an efficient scavenger, whereas, in B. subtilis, the relative numbers of mutations required for these two divergent evolutionary pathways would have been reversed. Other evidence also suggests that there is a fine balance between evolving organisms either to use unnatural amino acids or to avoid them. For example, a mutant E. coli selected for p-fluorophenylalanine resistance acquired a mutation in the phenylalanyl-tRNA synthetase (PheRS) that actually conferred greater discrimination against that amino-acid analogue [32]. Models for proteome evolution The best working hypothesis for the observed preference for 4fW in HR15 is that relatively few proteins were affected, and sites within those proteins now not only accommodate, but also strongly prefer the analogue. A strong prediction of this hypothesis is that HR15 should not grow well on other tryptophan analogues. In addition, the relative refractivity of the genetic code to change should be roughly dependent on the size of an organismal genome or proteome. A smaller proteome would have fewer functions and a smaller degree of interdependence than would a larger proteome, and would be correspondingly more amenable to manipulation. In this respect, a bacteriophage has recently been adapted to a mixture of natural and unnatural amino acids. Although the mixture was highly toxic to the phage (, 1/10 fitness), after 25 cycles of selection, , 40% of the fitness on the natural amino acid was recovered on the mixture of amino acids [33]. Furthermore, genetic code alterations occur more often in smaller genomes, such as those of mitochondria or chloroplasts [10,17]. Consistent with these speculations, it might be expected that mitochondrial proteins that have evolved to accommodate a different genetic code would no longer be functional if translated with the standard code.
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Anaerobic, 100 generations
Compromised ValRS editing function
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host translational machinery. However, ‘top down’ evolutionary approaches might still be required to facilitate substantially changes in amino-acid chemistry across an entire organism. The probable utility of combining the two approaches has recently been demonstrated by Schimmel, Marlie`re and co-workers. A selection for bacterial variants that insert an essential cysteine at an introduced valine codon in thymidilate synthase (thyA) yielded a mutant valyl-tRNA synthetase (ValRS) [50]. The editing domain responsible for hydrolyzing mischarged tRNAs was inactivated, enabling the accumulation of sufficient valyl-tRNA acylated with cysteine to promote growth (Figure 2). This selection for misincorporation of cysteine also led to an organism that could partially incorporate a-aminobutyrate throughout the bacterial proteome (24% of expected valine residues have incorporated a-aminobutyrate). Furthermore, a ‘top down’ approach led to the accumulation of
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Melding the methods Based on these results and interpretations, it seems likely that ‘bottom up’ engineering approaches can be used to bypass the considerable historical barriers imposed by the
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ut m
Rational engineering of the genetic code by ‘bottom up’ approaches During protein synthesis, canonical amino acids are matched with the appropriate SENSE CODONS through the specific recognition of amino acids by cognate aaRSs. CHARGED T RNAs, in turn, pair with mRNAs in the ribosome, ultimately resulting in the translation of mRNA into protein. Engineering an aaRS that can attach an unnatural amino acid to a SUPPRESSOR T RNA or to a cognate tRNA should therefore enable complete sitespecific incorporation of unnatural amino acids into a few proteins [34] or the partial incorporation into many proteins, respectively. The partial incorporation of unnatural amino acids throughout a proteome has been engineered by molecular manipulation and evolved by directed evolution. For example, variants of tyrosyl-tRNA synthetase (TyrRS) that could more readily incorporate azatyrosine were found by directly screening a library of TyrRS mutants for increased levels of incorporation of 3H-azatyrosine. The recovered F130S mutant is nearly twice as good at incorporating the analogue relative to the wild-type E. coli protein [35]. Tirrell and co-workers have mutated the editing site of a leucyl-tRNA synthetase and thereby facilitated the incorporation of a variety of leucine analogues into the E. coli proteome [36]. Similarly, the A294G substitution in phenylalanyl-tRNA synthetase (PheRS), was found to promote incorporation of p-fluoro-, p-chloro-, and p-bromophenylalanine [37 – 39]. Tirrell’s group has also ‘broken’ the degeneracy of the genetic code by engineering both a yeast PheRS and the corresponding tRNAPhe to better incorporate the analogue naphthylalanine across from some phenylalanine codons [40]. Highly effective methods for the in vivo incorporation of unnatural amino acids have been developed, but remain limited to site-specific incorporation (Box 2). In particular, stop codon suppression has now been shown to enable the substitution of specific protein residues with a wide range of tyrosine analogues [41 –47]. These results can be seen as experimental validation of the codon capture hypothesis, in which a codon (in this case an amber stop codon) can be reassigned to a new amino acid via a tRNA with a mutant anticodon sequence. The chemical diversity of the aminoacid substitutions introduced by codon capture methods will probably be much larger than the diversity that can be achieved via ambiguous intermediates, although the overall number of codons (and proteins) affected will be much smaller. The further refinement of codon capture methods and the development of expanded genetic codes will probably be driven by the biotechnological advantages that accrue to proteins that have expanded chemical functionalities. Indeed, the site-specific or whole-cell incorporation of unnatural amino acids has already been used to alter substantially the optical characteristics of fluorescent proteins [48,49].
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Figure 2. Selection of Escherichia coli variants that miscode cysteine for valine. Bacteria with an insertion in the chromosomal thyA gene were complemented by a thyA gene on a plasmid in which the crucial cysteine encoded at position 146 was changed to a valine. Bacteria were selected for growth in various ways. First, bacteria were selected for anaerobic growth over 100 generations in the presence of limiting cysteine. Four additional separate selections were carried out, starting from a derivative of the initial strain that contained a mutator phenotype. These latter selections were carried out on plates containing the cysteine analogue, S-carbamoylcysteine (Scc). One colony from each of the five selections was picked and characterized; mutations were found to co-transduce with a selectable marker near the gene encoding valyl-tRNA synthetase (valS). Each clone had a mutation in the editing domain of ValRS. These mutations hindered editing of mischarged tRNAs [50].
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Box 2. Bottom up approaches for unnatural amino-acid incorporation Site-specific incorporation of amino-acid analogues can be accomplished through the use of an orthogonal aminoacyl-tRNA synthetase (aaRS) and stop codon suppressor tRNA pairs. An orthogonal pair refers to a tRNA that is not charged by any of the host aaRSs, and an aaRS that does not charge any host tRNA (Figure I). Orthogonal pairs in Escherichia coli have been generated by rational design, by adapting tRNA:aaRS pairs from heterologous organisms, and by evolutionary engineering [54]. Unfortunately, rational design of an orthogonal system failed to result in a synthetase that could aminoacylate the engineered tRNA more efficiently than it did wild-type tRNA; in other words, this method did not create a fully orthogonal system [55,56]. To
remedy this problem, tRNA:aaRS pairs from other organisms were screened for orthogonality relative to the E. coli translation machinery [57]. These techniques were further developed to generate successfully fully orthogonal pairs and led to the adaptation of a TyrRS/tRNATyr orthogonal pair from Methanococcus janaschii to encode the unnatural amino acid O -methyl-L -tyrosine in E. coli [41,58,59]. In fact, O-methyl-L tyrosine was inserted into a single protein at the UAG stop codon in place of tyrosine at a fidelity close to that seen for the incorporation of natural amino acids [41]. A similar approach has been used to insert sitespecifically O -iodo-L -tyrosine into proteins in a cell-free, wheat germ eukaryotic translation system [60], as well as in a eukaryotic system [61].
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Figure I. Translation of the genetic code and adaptation of orthogonal aminoacyl-tRNA synthetase:tRNA pair for unnatural amino acid incorporation. The genetic code is deciphered in a three step process involving tRNAs, aminoacyl-tRNA synthetases, and the ribosome. In the first step (a), the aminoacyl-tRNA synthetase recognizes its cognate amino acid and forms a charged aminoacyl-adenylate in an ATP dependent process. Next (b), the aminoacyl-tRNA synthetase attaches the amino acid to the 30 adenosine of the cognate tRNA to form a ‘charged tRNA.’ Lastly (c), the charged tRNA is ready for translation of the genetic code at the ribosome where the amino acid is transferred from the tRNA to the nascent polypeptide chain (endogenous pair). This mechanism can be adapted to incorporate unnatural amino acids site-specifically without the insertion of any canonical amino acid through the use of an orthogonal aaRS:tRNA pair, thus altering the translation of the code (orthogonal pair). The amino-acid specificity of the aaRS can potentially be manipulated by design or selection, enabling different unnatural amino acids to be used in place of the natural amino-acid substrate [54].
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mutations in an aaRS that was equivalent to a mutation that might have been engineered by a ‘bottom up’ approach. In fact, strains containing a ValRS with a single editing mutation [50] were inhibited for growth by a-aminobutyrate in a rank order correlating to the severity of the mutation [51]. Conclusion and future prospects Biotechnology experiments provide evidence that historical modifications in the genetic code could potentially have occurred either via ambiguous intermediates or codon capture. These experiments also provide some additional insights into the parameters that would have affected codon evolution: proteome-wide substitutions that involved structurally similar amino acids might have occurred via ambiguous intermediates, whereas more limited substitutions that involve more chemically diverse amino acids may have occurred via codon capture. The former mechanism would seem to apply more naturally to most of the changes in the mitochondrial genetic code that have been observed, whereas the latter mechanism is probably how selenocysteine came to be encoded. Once a novel amino-acid substitution has been introduced into an organism, it might prove possible to adapt the organism more fully to utilize its novel amino-acid chemistry and new genetic code. Towards this end, a bacterium has been genetically engineered that not only incorporates the unnatural amino acid p-aminophenylalanine, but also synthesizes this compound from simpler chemical species [52]. The availability of new chemistries might provide an evolutionary advantage relative to organisms limited to a genetic code with the standard 20 amino acids. Studying the evolution of organisms that have been forced to incorporate unnatural amino acids might provide an excellent means to better understand genome and proteome evolution. Previous evolution experiments have yielded insights into a given protein or pathway, but few of these experiments disturbed the organism as a whole. By requiring coordinated mutational change throughout a large set of proteins, it might be possible to determine which portions of the genome and proteome are dependent upon, or interact with, one another. In other words, the disturbance and adaptation of the metabolic network as a whole provides a ready means to characterize and evaluate the interactions within that network. For example, if we had not already known that the tryptophanyl-tRNA synthetase, the aromatic amino acid permease and a repressor protein were all part of a common network before carrying out directed evolution experiments, it would have become apparent upon identifying these genetic changes in the organism evolved to incorporate 4-fluorotryptophan. To the extent that the genetic codes of organisms can be reformatted, it might be possible to revise Crick’s ‘frozen accident’ [1] and to remake chemical choices that have been fixed in place for billions of years. Ultimately, by changing the complement of amino acids that an organism uses, it should prove possible to change the basal chemistries available to that organism, and to open new options and avenues for evolution (e.g. the incorporation of a greater variety of non-polar, uncharged amino acids www.sciencedirect.com
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might enable the evolution of bacteria that would grow at temperatures even higher than those currently observed [53]). Moreover, by developing methods that enable the genetic code to float and evolve on its own, it might become possible to study the evolution of evolvability itself, as organisms adapt to competing codes representing a variety of chemical possibilities. Acknowledgements The authors thank Jim Bull, Jeff Tabor, David Metzgar and Vale´rie de Cre´cy-Lagard for critical reading of this article and their valuable suggestions. This research was supported by NASA and the Scripps Research Institute (Grant 5-97458). J.M.B. is currently at the Scripps Research Institute, 10550 N. Torrey Pines, BCC-379, La Jolla, CA, 92037, USA.
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24 Petrosino, J. et al. (1998) b-lactamases: protein evolution in real time. Trends Microbiol. 6, 323– 327 25 Mortlock, R.P. (1982) Metabolic acquisitions through laboratory selection. Annu. Rev. Microbiol. 36, 259– 284 26 Clarke, P.H. and Drew, R. (1988) An experiment in enzyme evolution. Studies with Pseudomonas aeruginosa amidase. Biosci. Rep. 8, 103–120 27 Wong, J.T.F. (1983) Membership mutation of the genetic code: loss of fitness by tryptophan. Proc. Natl. Acad. Sci. U. S. A. 80, 6303– 6306 28 Kunst, F. et al. (1997) The complete genome sequence of the grampositive bacterium Bacillus subtilis. Nature 390, 249– 256 29 Pratt, E.A. and Ho, C. (1975) Incorporation of fluorotryptophans into proteins of Escherichia coli. Biochemistry 14, 3035– 3040 30 Browne, D.R. et al. (1970) Incorporation of monoflurotryptophans into protein during the growth of Escherichia coli. Biochem. Biophys. Res. Commun. 39, 13– 19 31 Bacher, J.M. and Ellington, A.D. (2001) Selection and characterization of Escherichia coli variants capable of growth on an otherwise toxic tryptophan analogue. J. Bacteriol. 183, 5414– 5425 32 Hennecke, H. and Bock, A. (1975) Altered a subunits in phenylalanyltRNA synthetases from p-fluorophenylalanine-resistant strains of Escherichia coli. Eur. J. Biochem. 55, 431 – 437 33 Bacher, J.M. et al. Evolution of phage with chemically ambiguous proteomes. BMC Evol. Biol. (in press) 34 Dougherty, D.A. (2000) Unnatural amino acids as probes of protein structure and function. Curr. Opin. Chem. Biol. 4, 645 – 652 35 Hamano-Takaku, F. et al. (2000) A mutant Escherichia coli tyrosyltRNA synthetase utilizes the unnatural amino acid azatyrosine more efficiently than tyrosine. J. Biol. Chem. 275, 40324 – 40328 36 Yang, Y. and Tirrell, D.A. (2002) Attenuation of the editing activity of the Escherichia coli leucyl-tRNA synthetase allows incorporation of novel amino acids into proteins in vivo. Biochemistry 41, 10635 – 10645 37 Ibba, M. and Hennecke, H. (1995) Relaxing the substrate specificity of an aminoacyl-tRNA synthetase allows in vitro and in vivo synthesis of proteins containing unnatural amino acids. FEBS Lett. 364, 272– 275 38 Ibba, M. et al. (1994) Substrate specificity is determined by amino acid binding pocket size in Escherichia coli phenylalanyl-tRNA synthetase. Biochemistry 33, 7107– 7112 39 Kast, P. and Hennecke, H. (1991) Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations. J. Mol. Biol. 222, 99 – 124 40 Kwon, I. et al. (2003) Breaking the degeneracy of the genetic code. J. Am. Chem. Soc. 125, 7512– 7513 41 Wang, L. et al. (2001) Expanding the genetic code of Escherichia coli. Science 292, 498 – 500 42 Wang, L. et al. (2002) Adding L-3-(2-naphthyl)alanine to the genetic code of E. coli. J. Am. Chem. Soc. 124, 1836– 1837 43 Zhang, Z. et al. (2002) The selective incorporation of alkenes into proteins in Escherichia coli. Angew. Chem. Int. Ed. Engl. 41, 2840–2842
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44 Chin, J.W. et al. (2002) Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 99, 11020– 11024 45 Chin, J.W. et al. (2002) Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J. Am. Chem. Soc. 124, 9026– 9027 46 Chin, J.W. and Schultz, P.G. (2002) In vivo photocrosslinking with unnatural amino acid mutagenesis. Chembiochem 3, 1135– 1137 47 Santoro, S.W. et al. (2002) An efficient system for the evolution of aminoacyl-tRNA synthetase specificity. Nat. Biotechnol. 20, 1044– 1048 48 Bae, J.H. et al. (2003) Expansion of the genetic code enables design of a novel ‘gold’ class of green fluorescent proteins. J. Mol. Biol. 328, 1071– 1081 49 Wang, L. et al. (2003) Unnatural amino Acid mutagenesis of green fluorescent protein. J. Org. Chem. 68, 174 – 176 50 Do¨ring, V. et al. (2001) Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway. Science 292, 501 – 504 51 Nangle, L.A. et al. (2002) Genetic code ambiguity. Cell viability related to the severity of editing defects in mutant tRNA synthetases. J. Biol. Chem. 277, 45729 – 45733 52 Mehl, R.A. et al. (2003) Generation of a bacterium with a 21 amino acid genetic code. J. Am. Chem. Soc. 125, 935 – 939 53 Fields, P.A. (2001) Review: protein function at thermal extremes: balancing stability and flexibility. Comp. Biochem. Physiol. A 129, 417– 431 54 Wang, L. and Schultz, P.G. (2002) Expanding the genetic code. Chem. Commun. 1, 1 – 11 55 Liu, D.R. et al. (1997) Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo. Proc. Natl. Acad. Sci. U. S. A. 94, 10092 – 10097 56 Liu, D.R. et al. (1997) Characterization of an ‘orthogonal’ suppressor tRNA derived from E. coli tRNA2(Gln). Chem. Biol. 4, 685– 691 57 Liu, D.R. and Schultz, P.G. (1999) Progress toward the evolution of an organism with an expanded genetic code. Proc. Natl. Acad. Sci. U. S. A. 96, 4780 – 4785 58 Wang, L. et al. (2000) A new functional suppressor tRNA/aminoacyltRNA synthetase pair for the in vivo incorporation of unnatural amino acids into proteins. J. Am. Chem. Soc. 122, 5010– 5011 59 Wang, L. and Schultz, P.G. (2001) A general approach for the generation of orthogonal tRNAs. Chem. Biol. 8, 883 – 890 60 Kiga, D. et al. (2002) An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system. Proc. Natl. Acad. Sci. U. S. A. 99, 9715 – 9720 61 Sakamoto, K. et al. (2002) Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells. Nucleic Acids Res. 30, 4692– 4699
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Evolving new genetic codes Jamie M. Bacher1, Randall A. Hughes2, J. Tze-Fei Wong3 and Andrew D. Ellington1,2 1
Institute of Cellular and Molecular Biology, University of Texas at Austin, 1 University Station A4800, Austin, TX, 78712, USA Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A5300, Austin, TX, 78712, USA 3 Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong 2
Although the genetic code is almost universal, natural variations exist that have caused evolutionary biologists to speculate about codon evolution. There are two predominant hypotheses that specify either a gradual (ambiguous intermediate) or stochastic (codon capture) change in the code. These hypotheses are similar to two biotechnology techniques that have been used to engineer the genetic code: a ‘top down’ approach, in which the whole organism is evolved for the ability to incorporate unnatural amino acids, and a ‘bottom up’ approach, in which aminoacyl-tRNA synthetases and their cognate tRNAs are engineered. The biotechnology experiments provide insights into natural codon evolution, and a combination of these approaches should enable the evolution of organisms that can incorporate unnatural amino acids throughout their proteomes. Although the genetic code was discovered some 40-odd years ago, there are still numerous questions as to how it arose and evolved. One of the first and most famous hypotheses regarding the origin of the code was that it was a ‘frozen accident’ [1], in which associations between codons and amino acids were fixed in place and could change only with difficulty. Since Francis Crick proposed this theory, there have been several alternate explanations for the origins of the code, including stereochemical hypotheses that posit direct interactions between codons and COGNATE AMINO ACIDS (see Glossary) (see, for example [2]) and biosynthetic hypotheses that propose that the code was assigned in parallel to the evolution of amino-acid biosyntheses [3,4]. However, most inquiries have tended to focus on the establishment of the code, rather than on its alteration or maintenance. Although by and large, the code does remain ‘frozen,’ there are several examples of altered genetic codes in nature. One well characterized example is in mitochondrial genomes, where as many as 27 different codon reassignments have been observed [5]. The code can even be changed in free-living organisms; for example, in Candida albicans the ‘universal’ leucine codon CUG is decoded as serine rather than leucine [6]. In addition, novel amino acids, such as selenocysteine [7,8] and pyrrolysine [9], are sometimes incorporated at positions normally encoded by STOP CODONS . There are two principle hypotheses for how natural changes in the code might have evolved: the ‘ambiguous
intermediate’ and ‘codon capture’ hypotheses (reviewed in [10]). In the ‘ambiguous intermediate’ hypothesis [11 – 13], a duplicated and mutated tRNA recognizes a normally NON-COGNATE CODON and inserts its amino acid in competition with the cognate amino acid. Propagation of organisms with ambiguous proteomes could occur if the NON-COGNATE AMINO ACID were either close to selectively neutral or provided a net selective advantage that overcame any deficits in the function of individual proteins. The further evolution of those proteins whose functions were compromised by amino-acid substitutions would eventually repair any minor decreases in fitness. Following the adaptation of individual proteins, a discrete but altered genetic code could be re-established. By contrast, the ‘codon capture’ or ‘disappearing intermediate’ hypothesis [14– 16] posits that certain codons were eliminated by genetic drift throughout genomes that evolved skewed GC or AT contents. Following codon loss, relevant tRNA adaptors became functionless and were deleted. At some later point in evolution, sequence composition changed again, and a different tRNA Glossary Aminoacyl-tRNA synthetase (aaRS): the general name for the group of enzymes responsible for forming the covalent linkage between a specific amino acid and a specific tRNA molecule. Canonical amino acid: an amino acid that is part of the usual complement of 20 amino acids encoded by the genetic code. Charged tRNA: a tRNA that has been covalently linked to an amino acid. Cognate amino acid: an amino acid that is the natural substrate for a particular aminoacyl-tRNA synthetase and corresponds to specific codon(s) in the genetic code. For example, tryptophan is the cognate amino acid of tryptophanyl-tRNA synthetase and has one codon, UGG. Cognate codon: one of 61 nucleotide triplets that encodes for a specific amino acid according to the standard genetic code. For example, UGG is the cognate codon for tryptophan. Directed evolution: a process by which molecules or organisms are subjected to successive cycles of mutation and selection result ingin some desired improved functionality; also known as evolutionary engineering. Non-cognate amino acid: an amino acid that is not the natural substrate for a particular aminoacyl-tRNA synthetase and therefore does not naturally correspond to the codons associated with that amino acid. For example, serine is a non-cognate amino acid of leucyl-tRNA synthetase and, therefore, does not naturally correspond to any of the six codons of leucine. Non-cognate codon: a codon that is not normally assigned to a particular amino acid. For example, the leucine codon CUG is a non-cognate codon to serine. Refractivity: the ability to accept changes; flexibility. Sense codon: a codon that encodes a wild-type amino acid within a gene. Stop codon: a codon that results in termination of an expanding protein. Suppressor tRNA: a tRNA that has been genetically modified to recognize a stop codon; these will function to insert an amino acid at positions that would normally cause an end in protein synthesis. Unnatural amino acid: an amino acid that is not normally encoded by the genetic code; also referred to as an amino-acid analogue.
Corresponding author: Andrew D. Ellington ([email protected]). www.sciencedirect.com 0169-5347/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tree.2003.11.007
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adaptor duplicated, mutated at the anticodon position, and recaptured the codon that had previously disappeared. A variant of this hypothesis suggests that evolutionary pressure on several genotypic characteristics, including genome size and organization as well as composition, influenced codon reassignment [17]. Until recently, very little experimental evidence has been available regarding how mechanistically difficult it might be to reassign amino acids, what the consequences to organismal fitness of such reassignments might be, and how answers to these questions might bear on hypotheses regarding the evolution of the code. Nonetheless, experimental evolution is precisely the tool that should be used to probe these issues, and it has long been used to test theories of evolutionary biology. The combination of molecular biology and experimental evolution was initiated by Spiegelman and colleagues, who developed techniques to amplify the Qb genome entirely in vitro, and then demonstrated how selection pressures, such as faster replication or the introduction of ethidium bromide, could result in the fixation of mutations [18,19]. Furthermore, microorganisms have been evolved over large numbers of generations and in large populations to optimize growth rate [20,21], or to study the evolution of sex [22,23]. With the appropriate restriction of selection pressure, organismal evolution can also be used to evaluate the evolution of single enzymes, such as antibiotic resistance elements [24], or of metabolic pathways [25,26]. As such, microorganisms are effective model systems for the experimental evolution of a broad range of characteristics. Recently, several biotechnology experiments have begun to address these questions. The incorporation of UNNATURAL AMINO ACIDS into organismal proteomes has been achieved by appropriate genetic engineering and DIRECTED EVOLUTION of enzymes specifically involved in the translational apparatus, such as AMINOACYL-TRNA SYNTHETASES (aaRSs). Two different approaches have been used that, for simplicity, can be termed ‘top down’ and ‘bottom up.’ The ‘top down’ approach corresponds roughly to the ‘ambiguous intermediate’ hypothesis, whereas the ‘bottom up’ approach is more consistent with the ‘codon capture’ hypothesis (Box 1). Evolutionary engineering of the genetic code by ‘top down’ approaches Simple selection techniques were sufficient to induce a tryptophan auxotroph of Bacillus subtilis to grow when supplied with the amino acid 4-fluorotryptophan (4fW) in place of the CANONICAL AMINO ACID tryptophan (W; Figure 1) [27]. The chemical substitution of fluorine for hydrogen at the 4 position of W does not drastically alter the size or shape of the amino acid, although its polarity is changed substantially. Moreover, W is the most infrequently utilized amino acid, typically comprising , 1% of an organismal proteome. Even so, the genomic sequence of B. subtilis predicts that there are , 12 600 tryptophans spread over 4100 proteins [28]. It is likely that the functions of at least some of these proteins were critically affected, because studies with proteins that were individually substituted with fluorotryptophan have shown that enzymatic activities could be depressed by as much as an order of magnitude [29,30]. www.sciencedirect.com
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Box 1. The big picture † For the most part, the genetic code has remained ‘frozen’ or universally conserved throughout all taxa; however, a few naturally occurring variations have led to speculation about codon evolution. † There are two principle hypotheses for how natural changes in the code might have evolved: the ‘ambiguous intermediate’ hypothesis and the ‘codon capture’ hypothesis. † Experimental evolution can provide insights into how amino acids can be reassigned and what the effects are of such reassignments on organismal fitness. These experiments also provide additional insight into what biochemical parameters might affect codon evolution. † The ability to alter the genetic code is, in itself, a remarkable feat. The expansion of the chemical functionalities available to an organism should open new options and avenues for evolution, which will enable novel proteins and pathways to be constructed, as well as to expand greatly our understanding of evolutionary processes.
The parental strain of B. subtilis was plated on 4fW and colonies that were uniformly larger were picked. These variants were then mutagenized, and colonies that grew well in the presence of fluorotryptophan but poorly in the presence of tryptophan were picked. After four rounds of coupled positive and negative screening, clone HR15 was found that preferred 4fW for growth, by a ratio of 28:1 based on radioisotope incorporation; this represents a . 2 £ 104 switch in preference between the naturally occurring amino acid and the analogue. Amino-acid analysis revealed that HR15 completely incorporated the analogue into its proteome [27]. The REFRACTIVITY of the genetic code throughout its evolutionary history appears to be wildly at odds with the ease with which it can be manipulated experimentally. Although the initial strain showed limited growth in the presence of the unnatural amino acid, the apparent simplicity of this selection suggests that 4fW had a relatively minor impact on the function of the B. subtilis proteome. Based on these results, it appears that an organism with an ambiguous code can survive and perhaps even prosper if the noncognate amino acid does not greatly impair the function of crucial or numerous proteins. This might imply that ambiguous codon reassignments will be limited, in general, to either structurally similar amino acids or to codons that are used infrequently.
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Figure 1. Structures of tryptophan (a) and 4-fluorotryptophan (b). Tryptophan is shown with the 4, 5, and 6 positions indicated.
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To the extent that organismal fitness is impaired by codon ambiguity, there might be substantial resistance to altering the genetic code, as was found when a set of experiments designed to replace tryptophan with 4fW was performed with Escherichia coli [31]. In these experiments, a tryptophan auxotroph was grown in liquid media in the presence of increasingly higher ratios of 4fW:W, until only 4fW was added at the conclusion of the directed evolution experiment. Several mutations were identified, including changes in tryptophanyl-tRNA synthetase (trpS), the aromatic amino acid permease (aroP) and a repressor of AroP synthesis. However, in contrast to HR15, the evolved E. coli strain did not prefer 4fW for growth, and actually grew extremely poorly on 4fW alone. Although only 4fW was present in the evolved proteome at the limit of the detection methods used (,1% W would have been observed), it appeared that the evolved E. coli still required W for growth. The evolved E. coli had apparently developed the ability to scavenge the very low amounts of W that were present in the medium (commercial 4fW is contaminated with 0.03% W). The level of contaminating W might have been enough to enable the occasional production of proteins containing essential W residues and to therefore support only exceptionally slow growth. Evolutionary paths to amino-acid substitution The relatively small changes in amino-acid preference that were seen with E. coli were surprising, given the earlier results obtained using B. subtilis. The reason for this difference might be related to the number of proteins that would have been critically affected in each organism by the incorporation of the unnatural amino acid. Several lines of evidence indicate that the number might have been relatively small in the experiments with B. subtilis. First, the selection was carried out using a defined but exceptionally rich media that contained nucleotides, vitamins, cofactors and all of the canonical amino acids except W (as well as 4fW). Therefore, to the extent that 4fW incorporation would have compromised enzymes in many biosynthetic pathways, these deficiencies would have gone largely unnoticed. Second, HR15 was derived in only four rounds of colony screening coupled with a relatively low mutation rate and small population size, suggesting that relatively few W residues were critically impaired by replacement with 4fW. Thus, for most of the B. subtilis organismal proteome, the presence of W or 4fW was a largely neutral trait. Furthermore, the ancestral B. subtilis were capable of forming small colonies on the amino-acid analogue. As such, a new ability did not need to be evolved; instead, the selective regime was a higher growth rate under specific conditions. Finally, a selection against growth on W was performed on the B. subtilis variants, further encouraging growth on 4fW by limiting evolutionary options. This ‘negative selection’ might have been extremely important in the successful selection of HR15. By contrast, E. coli was evolved in a minimal media supplemented with 4fW and, thus, many more biosynthetic proteins would have been under selective pressure. In addition, the use of a chemical mutagen during the derivation of HR15 might have provided access to more www.sciencedirect.com
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diverse mutations than were available to the E. coli population. The one relative evolutionary advantage during the E. coli selection was a larger population size (, 107 E. coli serial dilution21 versus . 10 000 B. subtilis plated). The differences between the evolutionary paths taken by the two strains can also be attributed to other factors. The E. coli were evolved in liquid media rather than on plates. It would have therefore been more difficult for individual advantageous mutants to establish themselves because of competition among individuals compared with the direct selection by spatial segregation on plates to which B. subtilis was subjected. Nonetheless, some of the mutations that arose in the evolving E. coli lineage ‘swept’ through a given population within one cycle of serial dilution and growth. In addition, because diffusion was much greater in liquid than on solid media, scavenging strategies would have been more favored in the liquid environment. Finally, no negative selection against growth on W was applied to the evolved E. coli and, thus, robust growth would always have been the most probable option whenever sufficient W was present. Overall, the number of crucial mutations that were required during the E. coli selection to alter amino-acid preference was probably larger than the number that would have been required to generate an efficient scavenger, whereas, in B. subtilis, the relative numbers of mutations required for these two divergent evolutionary pathways would have been reversed. Other evidence also suggests that there is a fine balance between evolving organisms either to use unnatural amino acids or to avoid them. For example, a mutant E. coli selected for p-fluorophenylalanine resistance acquired a mutation in the phenylalanyl-tRNA synthetase (PheRS) that actually conferred greater discrimination against that amino-acid analogue [32]. Models for proteome evolution The best working hypothesis for the observed preference for 4fW in HR15 is that relatively few proteins were affected, and sites within those proteins now not only accommodate, but also strongly prefer the analogue. A strong prediction of this hypothesis is that HR15 should not grow well on other tryptophan analogues. In addition, the relative refractivity of the genetic code to change should be roughly dependent on the size of an organismal genome or proteome. A smaller proteome would have fewer functions and a smaller degree of interdependence than would a larger proteome, and would be correspondingly more amenable to manipulation. In this respect, a bacteriophage has recently been adapted to a mixture of natural and unnatural amino acids. Although the mixture was highly toxic to the phage (, 1/10 fitness), after 25 cycles of selection, , 40% of the fitness on the natural amino acid was recovered on the mixture of amino acids [33]. Furthermore, genetic code alterations occur more often in smaller genomes, such as those of mitochondria or chloroplasts [10,17]. Consistent with these speculations, it might be expected that mitochondrial proteins that have evolved to accommodate a different genetic code would no longer be functional if translated with the standard code.
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Anaerobic, 100 generations
Compromised ValRS editing function
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host translational machinery. However, ‘top down’ evolutionary approaches might still be required to facilitate substantially changes in amino-acid chemistry across an entire organism. The probable utility of combining the two approaches has recently been demonstrated by Schimmel, Marlie`re and co-workers. A selection for bacterial variants that insert an essential cysteine at an introduced valine codon in thymidilate synthase (thyA) yielded a mutant valyl-tRNA synthetase (ValRS) [50]. The editing domain responsible for hydrolyzing mischarged tRNAs was inactivated, enabling the accumulation of sufficient valyl-tRNA acylated with cysteine to promote growth (Figure 2). This selection for misincorporation of cysteine also led to an organism that could partially incorporate a-aminobutyrate throughout the bacterial proteome (24% of expected valine residues have incorporated a-aminobutyrate). Furthermore, a ‘top down’ approach led to the accumulation of
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Rational engineering of the genetic code by ‘bottom up’ approaches During protein synthesis, canonical amino acids are matched with the appropriate SENSE CODONS through the specific recognition of amino acids by cognate aaRSs. CHARGED T RNAs, in turn, pair with mRNAs in the ribosome, ultimately resulting in the translation of mRNA into protein. Engineering an aaRS that can attach an unnatural amino acid to a SUPPRESSOR T RNA or to a cognate tRNA should therefore enable complete sitespecific incorporation of unnatural amino acids into a few proteins [34] or the partial incorporation into many proteins, respectively. The partial incorporation of unnatural amino acids throughout a proteome has been engineered by molecular manipulation and evolved by directed evolution. For example, variants of tyrosyl-tRNA synthetase (TyrRS) that could more readily incorporate azatyrosine were found by directly screening a library of TyrRS mutants for increased levels of incorporation of 3H-azatyrosine. The recovered F130S mutant is nearly twice as good at incorporating the analogue relative to the wild-type E. coli protein [35]. Tirrell and co-workers have mutated the editing site of a leucyl-tRNA synthetase and thereby facilitated the incorporation of a variety of leucine analogues into the E. coli proteome [36]. Similarly, the A294G substitution in phenylalanyl-tRNA synthetase (PheRS), was found to promote incorporation of p-fluoro-, p-chloro-, and p-bromophenylalanine [37 – 39]. Tirrell’s group has also ‘broken’ the degeneracy of the genetic code by engineering both a yeast PheRS and the corresponding tRNAPhe to better incorporate the analogue naphthylalanine across from some phenylalanine codons [40]. Highly effective methods for the in vivo incorporation of unnatural amino acids have been developed, but remain limited to site-specific incorporation (Box 2). In particular, stop codon suppression has now been shown to enable the substitution of specific protein residues with a wide range of tyrosine analogues [41 –47]. These results can be seen as experimental validation of the codon capture hypothesis, in which a codon (in this case an amber stop codon) can be reassigned to a new amino acid via a tRNA with a mutant anticodon sequence. The chemical diversity of the aminoacid substitutions introduced by codon capture methods will probably be much larger than the diversity that can be achieved via ambiguous intermediates, although the overall number of codons (and proteins) affected will be much smaller. The further refinement of codon capture methods and the development of expanded genetic codes will probably be driven by the biotechnological advantages that accrue to proteins that have expanded chemical functionalities. Indeed, the site-specific or whole-cell incorporation of unnatural amino acids has already been used to alter substantially the optical characteristics of fluorescent proteins [48,49].
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Figure 2. Selection of Escherichia coli variants that miscode cysteine for valine. Bacteria with an insertion in the chromosomal thyA gene were complemented by a thyA gene on a plasmid in which the crucial cysteine encoded at position 146 was changed to a valine. Bacteria were selected for growth in various ways. First, bacteria were selected for anaerobic growth over 100 generations in the presence of limiting cysteine. Four additional separate selections were carried out, starting from a derivative of the initial strain that contained a mutator phenotype. These latter selections were carried out on plates containing the cysteine analogue, S-carbamoylcysteine (Scc). One colony from each of the five selections was picked and characterized; mutations were found to co-transduce with a selectable marker near the gene encoding valyl-tRNA synthetase (valS). Each clone had a mutation in the editing domain of ValRS. These mutations hindered editing of mischarged tRNAs [50].
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Box 2. Bottom up approaches for unnatural amino-acid incorporation Site-specific incorporation of amino-acid analogues can be accomplished through the use of an orthogonal aminoacyl-tRNA synthetase (aaRS) and stop codon suppressor tRNA pairs. An orthogonal pair refers to a tRNA that is not charged by any of the host aaRSs, and an aaRS that does not charge any host tRNA (Figure I). Orthogonal pairs in Escherichia coli have been generated by rational design, by adapting tRNA:aaRS pairs from heterologous organisms, and by evolutionary engineering [54]. Unfortunately, rational design of an orthogonal system failed to result in a synthetase that could aminoacylate the engineered tRNA more efficiently than it did wild-type tRNA; in other words, this method did not create a fully orthogonal system [55,56]. To
remedy this problem, tRNA:aaRS pairs from other organisms were screened for orthogonality relative to the E. coli translation machinery [57]. These techniques were further developed to generate successfully fully orthogonal pairs and led to the adaptation of a TyrRS/tRNATyr orthogonal pair from Methanococcus janaschii to encode the unnatural amino acid O -methyl-L -tyrosine in E. coli [41,58,59]. In fact, O-methyl-L tyrosine was inserted into a single protein at the UAG stop codon in place of tyrosine at a fidelity close to that seen for the incorporation of natural amino acids [41]. A similar approach has been used to insert sitespecifically O -iodo-L -tyrosine into proteins in a cell-free, wheat germ eukaryotic translation system [60], as well as in a eukaryotic system [61].
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Figure I. Translation of the genetic code and adaptation of orthogonal aminoacyl-tRNA synthetase:tRNA pair for unnatural amino acid incorporation. The genetic code is deciphered in a three step process involving tRNAs, aminoacyl-tRNA synthetases, and the ribosome. In the first step (a), the aminoacyl-tRNA synthetase recognizes its cognate amino acid and forms a charged aminoacyl-adenylate in an ATP dependent process. Next (b), the aminoacyl-tRNA synthetase attaches the amino acid to the 30 adenosine of the cognate tRNA to form a ‘charged tRNA.’ Lastly (c), the charged tRNA is ready for translation of the genetic code at the ribosome where the amino acid is transferred from the tRNA to the nascent polypeptide chain (endogenous pair). This mechanism can be adapted to incorporate unnatural amino acids site-specifically without the insertion of any canonical amino acid through the use of an orthogonal aaRS:tRNA pair, thus altering the translation of the code (orthogonal pair). The amino-acid specificity of the aaRS can potentially be manipulated by design or selection, enabling different unnatural amino acids to be used in place of the natural amino-acid substrate [54].
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mutations in an aaRS that was equivalent to a mutation that might have been engineered by a ‘bottom up’ approach. In fact, strains containing a ValRS with a single editing mutation [50] were inhibited for growth by a-aminobutyrate in a rank order correlating to the severity of the mutation [51]. Conclusion and future prospects Biotechnology experiments provide evidence that historical modifications in the genetic code could potentially have occurred either via ambiguous intermediates or codon capture. These experiments also provide some additional insights into the parameters that would have affected codon evolution: proteome-wide substitutions that involved structurally similar amino acids might have occurred via ambiguous intermediates, whereas more limited substitutions that involve more chemically diverse amino acids may have occurred via codon capture. The former mechanism would seem to apply more naturally to most of the changes in the mitochondrial genetic code that have been observed, whereas the latter mechanism is probably how selenocysteine came to be encoded. Once a novel amino-acid substitution has been introduced into an organism, it might prove possible to adapt the organism more fully to utilize its novel amino-acid chemistry and new genetic code. Towards this end, a bacterium has been genetically engineered that not only incorporates the unnatural amino acid p-aminophenylalanine, but also synthesizes this compound from simpler chemical species [52]. The availability of new chemistries might provide an evolutionary advantage relative to organisms limited to a genetic code with the standard 20 amino acids. Studying the evolution of organisms that have been forced to incorporate unnatural amino acids might provide an excellent means to better understand genome and proteome evolution. Previous evolution experiments have yielded insights into a given protein or pathway, but few of these experiments disturbed the organism as a whole. By requiring coordinated mutational change throughout a large set of proteins, it might be possible to determine which portions of the genome and proteome are dependent upon, or interact with, one another. In other words, the disturbance and adaptation of the metabolic network as a whole provides a ready means to characterize and evaluate the interactions within that network. For example, if we had not already known that the tryptophanyl-tRNA synthetase, the aromatic amino acid permease and a repressor protein were all part of a common network before carrying out directed evolution experiments, it would have become apparent upon identifying these genetic changes in the organism evolved to incorporate 4-fluorotryptophan. To the extent that the genetic codes of organisms can be reformatted, it might be possible to revise Crick’s ‘frozen accident’ [1] and to remake chemical choices that have been fixed in place for billions of years. Ultimately, by changing the complement of amino acids that an organism uses, it should prove possible to change the basal chemistries available to that organism, and to open new options and avenues for evolution (e.g. the incorporation of a greater variety of non-polar, uncharged amino acids www.sciencedirect.com
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might enable the evolution of bacteria that would grow at temperatures even higher than those currently observed [53]). Moreover, by developing methods that enable the genetic code to float and evolve on its own, it might become possible to study the evolution of evolvability itself, as organisms adapt to competing codes representing a variety of chemical possibilities. Acknowledgements The authors thank Jim Bull, Jeff Tabor, David Metzgar and Vale´rie de Cre´cy-Lagard for critical reading of this article and their valuable suggestions. This research was supported by NASA and the Scripps Research Institute (Grant 5-97458). J.M.B. is currently at the Scripps Research Institute, 10550 N. Torrey Pines, BCC-379, La Jolla, CA, 92037, USA.
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