Perspective Cite This: Biochemistry XXXX, XXX, XXX−XXX
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The Role of tRNA in Establishing New Genetic Codes Ross Thyer* and Andrew D. Ellington*
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Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, United States ABSTRACT: One of the most remarkable, but typically unremarked, aspects of the translation apparatus is the pleiotropic pliability of tRNA. This humble cloverleaf/Lshaped 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 noncognate 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 and 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.
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altering interactions with the web of endogenous synthetases.8 Similarly, the Saccharomyces cerevisiae tryptophanyl-tRNA synthetase:tRNA pair required substantial engineering of SctRNATrpCUA to reduce the extent of recognition by the endogenous lysyl-tRNA synthetase in E. coli. This was accomplished by systematically removing identity elements shared between 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. To improve activity, orthogonal tRNAs (Figure 1) can be adapted for specific interactions with their cognate aminoacyltRNA 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 that contained novel identity elements in other locations were identified.13 In
ince 2001, the genetic code has been engineered to encode more than 200 new amino acids with novel functional properties,1 including bioorthogonal labeling, metal chelation, and site specific post-translational modification, via orthogonal recoding strategies (see ref 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 incorporation of amino acids into proteins, their customization for novel noncanonical amino acid chemistries and more broadly their role in the development of new genetic codes remain underexplored. In broad terms, 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 finetuning association with other translational machinery. The original orthogonal translation system utilized the tyrosyl-tRNA synthetase:tRNA pair from the methanogenic archaeon Methanocaldococcus jannaschii5 and suffered from poor activity and significant toxicity in heterologous bacterial hosts. The 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 hypothesis that reduced fitness in Escherichia coli was a systemic effect, these improved variants have not only drastically improved activity but also decreased the level of background charging with canonical amino acids, possibly by © XXXX American Chemical Society
Special Issue: The Chemistry of Synthetic Biology Received: August 8, 2018 Revised: October 31, 2018 Published: November 2, 2018 A
DOI: 10.1021/acs.biochem.8b00834 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 1. Common orthogonal tRNAs used for genetic code expansion. (a) Tyrosine tRNA from the methanogenic archaeon M. jannaschii. (b) Pyrrolysine tRNA from the methanogenic archaeon Me. mazei. (c) Selenocysteine tRNA from E. coli. (d) Tryptophan tRNA from the yeast S. cerevisiae. Bases colored blue represent changes to the anticodon loop that enable efficient suppression of UAG stop codons. Orange regions represent major identity determinants that are recognized by the cognate aminoacyl-tRNA synthetase (seryl-tRNA synthetase for tRNASec). The green bases in the D-loop and the 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.
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
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 that suppress the UAG stop B
DOI: 10.1021/acs.biochem.8b00834 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry 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 also almost all orthogonal tRNAs. In the case of SepRS, further selection of the tRNA binding region resulted in additional improvements, completing the evolution of a custom identity element to offset the loss of direct anticodon recognition. In addition to the derivation of orthogonal aminoacyl-tRNA synthetase:tRNA pairs from genetically distant organisms, it has also proven to be 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 Methanosarcina mazei tRNAPyl,15 as well as orthogonal pairs for incorporation of proline and leucine that 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 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 EF-Tu 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 Noncanonical amino acids, lacking any evolutionary optimization, disrupt this equilibrium and can suffer from 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 EFTu 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 tRNAPyl21 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 that natively recodes the amber (UAG) stop codon or the UGU cysteine codon23 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 analogues 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 antideterminants will have to be introduced, perhaps including anticodon base pairing between unnatural nucleotides, such as the NaM-TPT3 base pair that was recently used to incorporate noncanonical amino acids into proteins in E. coli.24 Regardless of the scale, highly engineered, bespoke tRNAs will play a critical role as expanded genetic codes undergo the transition from the laboratory to the wider bioeconomy.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Ross Thyer: 0000-0002-0356-5790 Author Contributions
R.T. and A.D.E. contributed equally to this work. Funding
Funding from the National Cancer Institute (Grant 5K99CA207870-02 to R.T.) and AFOSR (Grant FA9550-141-0089 to A.D.E.) is acknowledged. Notes
The authors declare the following competing financial interest(s): 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 work.
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REFERENCES
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DOI: 10.1021/acs.biochem.8b00834 Biochemistry XXXX, XXX, XXX−XXX
