Subscriber access provided by The University of Texas at El Paso (UTEP)
Perspective
The role of tRNA in Establishing New Genetic Codes Ross Thyer, and Andrew D. Ellington Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00834 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
The role of tRNA in Establishing New Genetic Codes Ross Thyer* and Andrew D. Ellington* Author Affiliations: Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX Email*:
[email protected] [email protected] One of the most remarkable, but typically unremarked, aspects of the translation apparatus is the pleiotropic pliability of tRNA. This humble cloverleaf/L-shaped molecule must implement the first genetic code, via base-pairing and wobble interactions, but is also largely responsible for the specificity of the second genetic code, the pairings between amino acids, tRNA synthetases, and tRNAs. Despite the overarching similarities between tRNAs, they must nonetheless be specifically recognized by cognate tRNA synthetases and largely rejected by non-cognate synthetases. Conversely, despite the differences between tRNAs that allow such discrimination, they must be uniformly accepted by the ribosome, in part via the machinations of the translation elongation factors, which work with a diverse coterie of tRNA-amino acid conjugates to balance binding and loading. While it is easy to ascribe both discrimination and acceptance to the individual proteins (synthetases, EF-Tu/eEF-1) that recognize tRNAs, there is a large body of evidence that suggests that the sequences, structures, and dynamics of tRNAs are instrumental in the choices these proteins make. Since 2001 the genetic code has been engineered to encode more than 200 new amino acids with novel functional properties(1) – including bioorthogonal labelling, metal chelation, and site specific post-translational modification – via orthogonal recoding strategies (see Dumas et. al. (2015)(2) for a comprehensive pictorial reference of noncanonical amino acids). While borrowing genetically distant aminoacyl-tRNA synthetase:tRNA pairs to capture stop codons is now commonplace in many model organisms, most efforts to expand the genetic code have focused on the development of aminoacyl-tRNA synthetases with altered substrate specificities or ambitious genomewide recoding projects(3, 4). Despite the primacy of tRNAs in directing the specificity of amino acid incorporation into proteins, their customization for novel non-canonical amino acid chemistries, and more broadly their role in the development of new genetic codes remains underexplored. Broadly speaking, efforts to improve the function of tRNAs for expanded genetic codes can be divided into three approaches: improving function in the new host, optimizing interactions with an orthogonal aminoacyl-tRNA synthetase, and fine-tuning association with other translational machinery. The original orthogonal translation system utilized the tyrosyl-tRNA synthetase:tRNA pair from the methanogenic archaeon Methanocaldococcus jannaschii(5), and suffered from poor activity and significant toxicity in heterologous bacterial hosts. Observed toxicity may have been due in part to promiscuous interactions between the tRNA and the host translational machinery, and several new iterations of suppressor tRNAs have now been derived for this system, mostly through directed evolution(6, 7). In keeping with the
ACS Paragon Plus Environment
Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
hypothesis that reduced fitness in E. coli was a systemic effect, these improved variants have not only drastically improved activity, but also lowered background charging with canonical amino acids, possibly by altering interactions with the web of endogenous synthetases(8). Similarly, the S. cerevisiae tryptophanyl-tRNA synthetase:tRNA pair required substantial engineering of Sc-tRNATrpCUA to reduce recognition by the endogenous lysyl-tRNA synthetase in E. coli. This was accomplished by systematically removing identity elements shared between the Sc-tRNATrpCUA and Ec-tRNALysUUU and increasing the rigidity of the anticodon stem to improve suppressor function(9, 10). Recently, tRNAPyl (pyrrolysine) variants from Methanosarcina, the preferred orthogonal translation system (OTS) in mammalian cells, have been improved by the substitution of stabilizing Watson-Crick base pairs and bases from human tRNAs that facilitate interactions with the mammalian translation apparatus(11). These tRNA variants were considerably more stable than the parental tRNAPyl, remaining partially folded even under denaturing conditions, and were found to attain a higher intracellular concentration, resulting in improved amber suppression. In order to improve activity, orthogonal tRNAs can be adapted for specific interactions with their cognate aminoacyl-tRNA synthetases or other modifying enzymes. A hybrid selenocysteinyl-tRNA, constructed by replacing the acceptor stem of E. coli tRNASer with that of tRNASec(12), lacked many of the canonical identity elements located in the D-arm for selenocysteine synthase (SelA), the enzyme responsible for catalyzing the direct conversion of serine to selenocysteine on tRNASec. Using an enzymatic assay to screen for mutants that were superior substrates for SelA, variant tRNAs were identified which contained novel identity elements in other locations(13). In contrast, SepRS, which charges the cysteine tRNA with phosphoserine during cysteine biosynthesis in certain methanogenic archaea, strongly recognizes the tRNA anticodon (GCA), and tRNACysCUA variants which suppress the UAG stop codon are poor substrates for the synthetase. Mutagenesis of the region surrounding the CUA anticodon followed by selection in the presence of SepRS identified tRNA variants with 10-fold greater activity(14). Optimization of the bases flanking the anticodon has been used as a general strategy to improve the activity of not only those tRNAs whose anticodon is recognized by their cognate synthetase, but almost all orthogonal tRNAs. In the case of SepRS, further selection of the tRNA binding region resulted in additional improvements, essentially evolving a custom identity element to offset the loss of direct anticodon recognition. In addition to deriving orthogonal aminoacyl-tRNA synthetase:tRNA pairs from genetically distant organisms, it has also proven possible to engineer pairs that have never existed in nature. These include the most widely used pyrrolysine OTS, a combination of the Methanosarcina barkeri PylRS and the more orthogonal M. mazei tRNAPyl(15), as well as orthogonal pairs for incorporation of proline and leucine which were derived by combinatorial screening of several different archaeal tRNAs and synthetases(16, 17). In addition to heterologous pairings, consensus orthogonal tRNAs generated by aligning multiple archaeal tRNA genes to capture the critical identity elements and minimize destabilizing features have also been used with success(18). While these synthetic metagenomics approaches are an attractive way to identify new orthogonal pairs, without
ACS Paragon Plus Environment
Page 2 of 8
Page 3 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
Figure 1. Common orthogonal tRNAs used for genetic code expansion. (a) Tyrosine tRNA from the methanogenic archaeon Methanocaldococcus jannaschii. (b) Pyrrolysine tRNA from the methanogenic archaeon Methanosarcina mazei. (c) Selenocysteine tRNA from Escherichia coli. (d) Tryptophan tRNA from the yeast Saccharomyces cerevisiae. Bases indicated in blue represent changes to the anticodon loop which enable efficient suppression of UAG stop codons. Orange regions represent major identity determinants which are recognized by the cognate aminoacyl-tRNA synthetase (seryl-tRNA synthetase for tRNASec). The green bases in D-loop and presence of an additional base pair in the acceptor stem of tRNASec are the major identity elements for selenocysteine synthase. Red bases represent key mutations in the named highly engineered tRNA variants.
ACS Paragon Plus Environment
Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
requiring a complete understanding of the identity elements, a dedicated effort to optimize the tRNA is critical to achieve efficient translation. Of course, tRNAs must also interact with the other translation factors to deliver their amino acid cargoes to the ribosome. In particular, interactions between charged tRNAs and EFTu are finely balanced: a weak interaction with an amino acid will be offset by a strong interaction with its cognate tRNA, and vice versa(19). Non-canonical amino acids, lacking any evolutionary optimization, disrupt this equilibrium which can lead to inefficient translation. While much of the interface between tRNAs and EF-Tu is formed by the tRNA backbone, the terminal base pair of the acceptor stem and the adjacent base pairs in the T-stem contribute significantly to the strength of the interaction(20). Fortunately, these positions are quite plastic and can be used to tailor the interaction with EF-Tu without seriously affecting the secondary structure of the tRNA. Through targeted mutagenesis of this region, a panel of different M. jannaschii tRNATyr variants has been customized for use with a series of structurally diverse aromatic amino acids(7). This approach has also been used to improve incorporation of N-ε-acetyllysine using tRNAPyl(21) and has even been used to adapt the E. coli selenocysteinyl-tRNA to work with the canonical elongation factor EF-Tu, rather than its own native selenocysteine-specific elongation factor, SelB,(22). While directed evolution has so far proven up to the task of creating and optimizing orthogonal pairs, increasingly powerful bioinformatics tools and ever lower sequencing costs should allow researchers to scan the global biome for structurally distinct phylogenies of translational machinery, or even rare exceptions to the genetic code. An example of the latter is the recent identification of bacterial selenocysteine incorporation machinery which natively recodes the amber (UAG) stop codon or the UGU cysteine codon(23), rather than the opal (UGA) stop codon normally used to encode the selenoproteome. If additional coding options are identified in metagenomic data, they can quickly become targets for code expansion in model organisms. While the current improvements to orthogonal tRNAs have undoubtedly assisted the expansion of the genetic code, there are significant barriers to be overcome as more exotic and ambitious recoding is attempted. Most unnatural amino acids to date have been structural analogs of a small subset of canonical amino acids, and have been incorporated using just a handful of orthogonal aminoacyl-tRNA synthetase:tRNA pairs. As a 22nd and 23rd amino acid are added to the mix, the derivation of each new orthogonal pair will require that each new tRNA have exquisite specificity for its partner synthetase. Indeed, it seems likely that to facilitate code expansion, and avoid host synthetase recognition, novel identity elements and anti-determinants will have to be introduced, perhaps including anticodon base-pairing between unnatural nucleotides, such as the NaM-TPT3 base pair which was recently used to incorporate non-canonical amino acids in E. coli(24). Regardless of the scale, highly engineered, bespoke tRNAs will play a critical role as expanded genetic codes transition from the laboratory into the wider bioeconomy. Acknowledgements: Funding from the NIH/NCI (5K99CA207870-02 to R.T.) and AFOSR (FA9550-14-1-0089 to A.D.E.) is acknowledged.
ACS Paragon Plus Environment
Page 4 of 8
Page 5 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
‡Author Contributions R. T. and A. D. E. contributed equally to this work. R.T. and A.D.E. have equity in GRO Biosciences, a company developing protein therapeutics, and receive royalties from licensing material described in this manuscript. References: 1. 2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Chin, J. W. (2017) Expanding and reprogramming the genetic code, Nature 550, 53. Dumas, A., Lercher, L., Spicer, C. D., and Davis, B. G. (2015) Designing logical codon reassignment - Expanding the chemistry in biology, Chemical science 6, 50-69. Lajoie, M. J., Rovner, A. J., Goodman, D. B., Aerni, H.-R., Haimovich, A. D., Kuznetsov, G., Mercer, J. A., Wang, H. H., Carr, P. A., Mosberg, J. A., Rohland, N., Schultz, P. G., Jacobson, J. M., Rinehart, J., Church, G. M., and Isaacs, F. J. (2013) Genomically recoded organisms expand biological functions, Science (New York, N.Y.) 342, 357-360. Mukai, T., Hoshi, H., Ohtake, K., Takahashi, M., Yamaguchi, A., Hayashi, A., Yokoyama, S., and Sakamoto, K. (2015) Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon, Scientific reports 5, 9699. Wang, L., Magliery, T. J., Liu, D. R., and Schultz, P. G. (2000) A New Functional Suppressor tRNA/Aminoacyl−tRNA Synthetase Pair for the in Vivo Incorporation of Unnatural Amino Acids into Proteins, Journal of the American Chemical Society 122, 5010-5011. Wang, L., and Schultz, P. G. (2001) A general approach for the generation of orthogonal tRNAs, Chemistry & biology 8, 883-890. Guo, J., Melançon, C. E., 3rd, Lee, H. S., Groff, D., and Schultz, P. G. (2009) Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids, Angewandte Chemie (International ed. in English) 48, 9148-9151. Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. (2010) An enhanced system for unnatural amino acid mutagenesis in E. coli, J Mol Biol 395, 361-374. Hughes, R. A., and Ellington, A. D. (2010) Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA, Nucleic Acids Res 38, 6813-6830. Ellefson, J. W., Meyer, A. J., Hughes, R. A., Cannon, J. R., Brodbelt, J. S., and Ellington, A. D. (2013) Directed evolution of genetic parts and circuits by compartmentalized partnered replication, Nature Biotechnology 32, 97. Serfling, R., Lorenz, C., Etzel, M., Schicht, G., Böttke, T., Mörl, M., and Coin, I. (2018) Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells, Nucleic Acids Research 46, 1-10. Aldag, C., Brocker, M. J., Hohn, M. J., Prat, L., Hammond, G., Plummer, A., and Soll, D. (2013) Rewiring translation for elongation factor Tu-dependent selenocysteine incorporation, Angewandte Chemie (International ed. in English) 52, 1441-1445. Miller, C., Bröcker, M. J., Prat, L., Ip, K., Chirathivat, N., Feiock, A., Veszprémi, M., and Söll, D. (2015) A synthetic tRNA for EF-Tu mediated selenocysteine incorporation in vivo and in vitro, FEBS letters 589, 2194-2199.
ACS Paragon Plus Environment
Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
14.
15. 16.
17. 18. 19. 20. 21. 22. 23. 24.
Rogerson, D. T., Sachdeva, A., Wang, K., Haq, T., Kazlauskaite, A., Hancock, S. M., Huguenin-Dezot, N., Muqit, M. M., Fry, A. M., Bayliss, R., and Chin, J. W. (2015) Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog, Nat Chem Biol 11, 496-503. Hancock, S. M., Uprety, R., Deiters, A., and Chin, J. W. (2010) Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair, J Am Chem Soc 132, 14819-14824. Chatterjee, A., Xiao, H., and Schultz, P. G. (2012) Evolution of multiple, mutually orthogonal prolyl-tRNA synthetase/tRNA pairs for unnatural amino acid mutagenesis in Escherichia coli
