aminoacyl-tRNA Synthetase Pairs Research Articles

Shedding light on ORAI1 channel with genetic code expansion

Genetic code expansion technology has been widely applied to control protein activity and biological systems by taking advantage of an amber stop codon suppressor tRNA and orthogonal aminoacyl-tRNA synthetase pair. With this chemical biology approach, Maltan et al. incorporated photocrosslinking unnatural amino acids (UAAs) into the transmembrane domains of ORAI1 to enable UV light-inducible calcium influx across the plasma membrane, mechanistic interrogation of the calcium release-activated calcium (CRAC) channel at the single amino acid level, and remote control of downstream calcium-modulated signaling in mammalian cells.

Synthetic Tyrosine tRNA Molecules with Noncanonical Secondary Structures.

The L-shape form of tRNA is maintained by tertiary interactions occurring in the core. Base changes in this domain can cause structural defects and impair tRNA activity. Here, we report on a method to safely engineer structural variations in this domain utilizing the noncanonical scaffold of tRNAPyl. First, we constructed a naïve hybrid between archaeal tRNAPyl and tRNATyr, which consisted of the acceptor and T stems of tRNATyr and the other parts of tRNAPyl. This hybrid tRNA efficiently translated the UAG codon to 3-iodotyrosine in Escherichia coli cells, when paired with a variant of the archaeal tyrosyl-tRNA synthetase. The amber suppression efficiency was slightly lower than that of the “bench-mark” archaeal tRNATyr suppressor assuming the canonical structure. After a series of modifications to this hybrid tRNA, we obtained two artificial types of tRNATyr: ZtRNA had an augmented D (auD) helix in a noncanonical form and the D and T loops bound by the standard tertiary base pairs, and YtRNA had a canonical auD helix and non-standard interloop interactions. It was then suggested that the ZtRNA scaffold could also support the glycylation and glutaminylation of tRNA. The synthetic diversity of tRNA would help create new tRNA–aminoacyl-tRNA synthetase pairs for reprogramming the genetic code.

Pyrrolysyl-tRNA Synthetase with a Unique Architecture Enhances the Availability of Lysine Derivatives in Synthetic Genetic Codes.

Genetic code expansion has largely relied on two types of the tRNA—aminoacyl-tRNA synthetase pairs. One involves pyrrolysyl-tRNA synthetase (PylRS), which is used to incorporate various lysine derivatives into proteins. The widely used PylRS from Methanosarcinaceae comprises two distinct domains while the bacterial molecules consist of two separate polypeptides. The recently identified PylRS from Candidatus Methanomethylophilus alvus (CMaPylRS) is a single-domain, one-polypeptide enzyme that belongs to a third category. In the present study, we showed that the PylRS—tRNAPyl pair from C. M. alvus can incorporate lysine derivatives much more efficiently (up to 14-times) than Methanosarcinaceae PylRSs in Escherichia coli cell-based and cell-free systems. Then we investigated the tRNA and amino-acid recognition by CMaPylRS. The cognate tRNAPyl has two structural idiosyncrasies: no connecting nucleotide between the acceptor and D stems and an additional nucleotide in the anticodon stem and it was found that these features are hardly recognized by CMaPylRS. Lastly, the Tyr126Ala and Met129Leu substitutions at the amino-acid binding pocket were shown to allow CMaPylRS to recognize various derivatives of the bulky Nε-benzyloxycarbonyl-l-lysine (ZLys). With the high incorporation efficiency and the amenability to engineering, CMaPylRS would enhance the availability of lysine derivatives in expanded codes.

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine (DOPA) and Its Application to Protein Conjugation.

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical properties, the amino acid has multiple uses in biochemical applications. This report describes a protocol for the genetic incorporation of biosynthesized DOPA and its application to protein conjugation. DOPA is biosynthesized by a tyrosine phenol-lyase (TPL) from catechol, pyruvate, and ammonia, and the amino acid is directly incorporated into proteins by the genetic incorporation method using an evolved aminoacyl-tRNA and aminoacyl-tRNA synthetase pair. This direct incorporation system efficiently incorporates DOPA with little incorporation of other natural amino acids and with better protein yield than the previous genetic incorporation system for DOPA. Protein conjugation with DOPA-containing proteins is efficient and site-specific and shows its usefulness for various applications. This protocol provides protein scientists with detailed procedures for the efficient biosynthesis of mutant proteins containing DOPA at desired sites and their conjugation for industrial and pharmaceutical applications.

Directed Evolution of Heterologous tRNAs Leads to Reduced Dependence on Post-transcriptional Modifications.

Heterologous tRNA:aminoacyl tRNA synthetase pairs are often employed for noncanonical amino acid incorporation in the quest for an expanded genetic code. In this work, we investigated one possible mechanism by which directed evolution can improve orthogonal behavior for a suite of Methanocaldococcus jannaschii ( Mj) tRNATyr-derived amber suppressor tRNAs. Northern blotting demonstrated that reduced expression of heterologous tRNA variants correlated with improved orthogonality. We suspected that reduced expression likely minimized nonorthogonal interactions with host cell machinery. Despite the known abundance of post-transcriptional modifications in tRNAs across all domains of life, few studies have investigated how host enzymes may affect behavior of heterologous tRNAs. Therefore, we measured tRNA orthogonality using a fluorescent reporter assay in several modification-deficient strains, demonstrating that heterologous tRNAs with high expression are strongly affected by some native E.coli RNA-modifying enzymes, whereas low abundance evolved heterologous tRNAs are less affected by these same enzymes. We employed mass spectrometry to map ms2i6A37 and Ψ39 in the anticodon arm of two high abundance tRNAs (Nap1 and tRNAOptCUA), which provides (to our knowledge) the first direct evidence that MiaA and TruA post-transcriptionally modify evolved heterologous amber suppressor tRNAs. Changes in total tRNA modification profiles were observed by mass spectrometry in cells hosting these and other evolved suppressor tRNAs, suggesting that the demonstrated interactions with host enzymes might disturb native tRNA modification networks. Together, these results suggest that heterologous tRNAs engineered for specialized amber suppression can evolve highly efficient suppression capacity within the native post-transcriptional modification landscape of host RNA processing machinery.

Expanding the genetic code in vertebrates.

Synthetic Biology Unnatural amino acids can be incorporated into target proteins by using a transfer RNA (tRNA)—aminoacyl-tRNA synthetase pair that adds an unnatural amino acid at an amber stop codon. The strategy has been used in prokaryotic and eukaryotic cells and for generating transgenic invertebrates. Chen et al. have succeeded in integrating a construct coding for the unnatural amino acid p -azido-phenylalanine (AzF) into the mouse genome. The artificial gene was transmitted to subsequent generations and caused no obvious physiological defects. Primary cells from adult transgenic mice incorporated AzF into a fluorescent reporter protein containing an amber codon. Similarly, the genetic code of zebrafish was expanded, and in vivo incorporation of AzF into reporter protein was achieved. This opens the potential to investigate the function of target proteins in living animals. Cell Res. 10.1038/cr.2016.145 (2016).

Rapid and Inexpensive Evaluation of Nonstandard Amino Acid Incorporation in Escherichia coli.

By introducing engineered tRNA and aminoacyl-tRNA synthetase pairs into an organism, its genetic code can be expanded to incorporate nonstandard amino acids (nsAAs). The performance of these orthogonal translation systems (OTSs) varies greatly, however, with respect to the efficiency and accuracy of decoding a reassigned codon as the nsAA. To enable rapid and systematic comparisons of these critical parameters, we developed a toolkit for characterizing any Escherichia coli OTS that reassigns the amber stop codon (TAG). It assesses OTS performance by comparing how the fluorescence of strains carrying plasmids encoding a fused RFP-GFP reading frame, either with or without an intervening TAG codon, depends on the presence of the nsAA. We used this kit to (1) examine nsAA incorporation by seven different OTSs, (2) optimize nsAA concentration in growth media, (3) define the polyspecificity of an OTS, and (4) characterize evolved variants of amberless E.coli with improved growth rates.

Recoded organisms engineered to depend on synthetic amino acids.

Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels, and chemicals1. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems2,3, which include bioremediation4 and probiotics5. Although safeguards have been designed to control cell growth by essential gene regulation6, inducible toxin switches7, and engineered auxotrophies8, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations9,10. Here, we describe the construction of a series of genomically recoded organisms (GROs)11 whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase (aaRS) pair into the chromosome of a GRO that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering (MAGE)12, we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harboring 3 TAG codons in conserved functional residues13 of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼1011 cells on solid media for seven days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches2,3,6-10. We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternate genetic codes that impart genetic isolation by impeding horizontal gene transfer11 and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.

Incorporation of Non-Canonical Amino Acids.

In this chapter we discuss the strengths, caveats and technical considerations of three approaches for reprogramming the chemical composition of selected amino acids within a membrane protein. In vivo nonsense suppression in the Xenopus laevis oocyte, evolved orthogonal tRNA and aminoacyl-tRNA synthetase pairs and protein ligation for biochemical production of semisynthetic proteins have been used successfully for ion channel and receptor studies. The level of difficulty for the application of each approach ranges from trivial to technically demanding, yet all have untapped potential in their application to membrane proteins.

Genetic Incorporation of a Phenanthroline-Containing Amino Acid in Escherichia coli

A simple and general method that selectively introduces metal binding sites into a protein can greatly increase the ability to design and biosynthesize artificial metalloproteins. Here, we report the incorporation of a phenanthroline-containing amino acid (Phen-Ala) into proteins in Escherichia coli by using the tRNACUA and tyrosyl aminoacyl-tRNA synthetase pair (BpyRS) from Methanococcus jannaschii, which was originally developed for a bipyridine-containing amino acid (Bpy-Ala). The incorporation efficiency of BpyRS for PhenAla was comparable to that for Bpy-Ala. Because of its high metal-binding ability and characteristic spectral properties, Phen-Ala can be a useful alternative to the existing metal-chelating amino acids for the design and synthesis of artificial metalloproteins.

Using E. coli-based cell-free protein synthesis to evaluate the kinetic performance of an orthogonal tRNA and aminoacyl-tRNA synthetase pair

Even though the orthogonal tRNA and aminoacyl-tRNA synthetase pairs derived from the archaeon Methanocaldococcus jannaschii have been used for many years for site-specific incorporation of non-natural amino acids (nnAAs) in Escherichiacoli, their kinetic parameters have not been evaluated. Here we use a cell-free protein synthesis (CFPS) system to control the concentrations of the orthogonal components in order to evaluate their performance while supporting synthesis of modified proteins (i.e. proteins with nnAAs). Titration experiments and estimates of turnover numbers suggest that the orthogonal synthetase is a very slow catalyst when compared to the native E. coli synthetases. The estimated kcat for the orthogonal synthetase specific to the nnAA p-propargyloxyphenylalanine (pPaF) is 5.4×10−5s−1. Thus, this catalyst may be the limiting factor for nnAA incorporation when using this approach. These titration experiments also resulted in the highest reported cell-free accumulation of two different modified proteins (450±20μg/ml CAT109pAzF and 428±2μg/ml sfGFP23pPaF) using the standard KC6 cell extract and either the PANOx SP or the inexpensive Glu NMP cell-free recipe.

Dynamics at Specific Sites in Proteins Studied by 2D IR Vibrational Echo Spectroscopy

The techniques of two-dimensional infrared (2D IR) spectroscopy enable the study of fluctuations of molecules and their environments on fast timescales. Characterization of specific sites in proteins requires the use of extrinsic vibration probes to alleviate the spectral complexity that hinders the application of IR spectroscopy with protein samples. Heme-bound CO has been a popular vibrational probe of protein active sites and, for example, was employed to characterize the involvement of dynamics in the specificity of cytochrome P450 using 2D IR spectroscopy. In efforts toward extending the 2D IR studies beyond heme proteins, an azidophenylalanine vibrational probe was introduced at a specific site within myoglobin using orthogonal transfer RNA (tRNA)/aminoacyl-tRNA synthetase pairs evolved to insert azidophenylalanine in response to an amber codon. The azidophenylalanine vibrational probe was placed within the heme pocket at Phe43, permitting investigation of one site in myoglobin via two different vibrational probes - the genetically introduced azidophenylalanine and heme bound CO. The use of two probes allowed assessment of the perturbation due to introduction of each and the measurement of the heme pocket dynamics via two perspectives, showing that the reported dynamics were reflective of the protein motions, and not intrinsic to a particular probe. This approach can be used in the future to characterize the dynamics at virtually any site throughout virtually any protein.

Genetically encoding unnatural amino acids in neural stem cells and optically reporting voltage-sensitive domain changes in differentiated neurons.

Although unnatural amino acids (Uaas) have been genetically encoded in bacterial, fungal, and mammalian cells using orthogonal transfer RNA (tRNA)/aminoacyl-tRNA synthetase pairs, applications of this method to a wider range of specialized cell types, such as stem cells, still face challenges. While relatively straightforward in stem cells, transient expression lacks sufficient temporal resolution to afford reasonable levels of Uaa incorporation and to allow for the study of the longer term differentiation process of stem cells. Moreover, Uaa incorporation may perturb differentiation. Here, we describe a lentiviral-based gene delivery method to stably incorporate Uaas into proteins expressed in neural stem cells, specifically HCN-A94 cells. The transduced cells differentiated into neural progenies in the same manner as the wild-type cells. By genetically incorporating a fluorescent Uaa into a voltage-dependent membrane lipid phosphatase, we show that this Uaa optically reports the conformational change of the voltage-sensitive domain in response to membrane depolarization. The method described here should be generally applicable to other stem cells and membrane proteins.

Utilizing elongation factor Tu mutagenesis to incorporate large unnatural amino acids in vivo

Site‐specific in vivo incorporation of unnatural amino acids (UAAs) via aminoacyl tRNA synthetase (RS) mutagenesis has been widely used in protein structure and function studies. While a large, diverse family of UAAs has been incorporated into proteins, aminoacyl tRNA synthetases for certain UAAs seem problematic to evolve or generate. We developed a reporter system to explore substrate specificity of current UAA RSs. Using these permissive RSs, structural limitations have been explored. The photocrosslinking UAAs anthraquinonylalanine and 3‐(3’‐fluorenyl‐9’‐oxo)‐alanine should be incorporated by permissive RSs. However, incorporation of these UAAs into proteins has not yet been accomplished. It is the current understanding that elongation factor Tu (EF‐Tu) size limitation is preventing the incorporation of these UAAs. It is believed that UAAs that were previously too large to see incorporation with mutant RSs alone will hopefully be incorporated utilizing mutant RS and mutant EF‐Tu combinations in vivo. Here we report progress with mutant EF‐Tu and orthogonal aminoacyl tRNA synthetase pairs. Supported by F&M funds, NSF‐MCB‐0448297; Research Corporation (CC6364).

Novel proteins, novel amino acids

Expanding the limited set of 20 amino acids may lead to the generation of proteins with novel properties. Two papers published in the 20 April edition of Science indicate how to accomplish the incorporation of novel amino acids into proteins. The paper by Volker Döring et al., describes the production of mutants of Escherichia coli that incorrectly charge tRNA–valine with cysteine. More than 20% of the valine in cellular proteins in such a mutant organism could be replaced with another amino acid, aminobutyrate, which is similar in shape to cysteine, but is not used in the construction of proteins. The other paper, by Lei Wang et al., describes the construction of a unique tRNA–aminoacyl-tRNA synthetase pair, that, when introduced into E. coli, leads to the in vivo incorporation of the synthetic amino acid O–methyl–l–tyrosine. Using this approach, it is possible to introduce novel amino acids into proteins at any position of interest. DM

Expanding the genetic code of Escherichia coli.

A unique transfer RNA (tRNA)/aminoacyl-tRNA synthetase pair has been generated that expands the number of genetically encoded amino acids in Escherichia coli. When introduced into E. coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-l-tyrosine into protein in response to an amber nonsense codon. The fidelity of translation is greater than 99%, as determined by analysis of dihydrofolate reductase containing the unnatural amino acid. This approach should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chemical, and physical properties not found in the common 20 amino acids.