ArcS from Thermococcus kodakarensis transfers L-lysine to preQ0 nucleoside derivatives as minimum substrate RNAs.

Archaeosine (G+) is an archaea-specific tRNA modification synthesized via multiple steps. In the first step, archaeosine tRNA guanine transglucosylase (ArcTGT) exchanges the G15 base in tRNA with 7-cyano-7-deazaguanine (preQ0). In Euryarchaea, preQ015 in tRNA is further modified by archaeosine synthase (ArcS). Thermococcus kodakarensis ArcS catalyzes a lysine-transfer reaction to produce preQ0-lysine (preQ0-Lys) as an intermediate. The resulting preQ0-Lys15 in tRNA is converted to G+15 by a radical S-adenosyl-L-methionine enzyme for archaeosine formation (RaSEA), which forms a complex with ArcS. Here, we focus on the substrate tRNA recognition mechanism of ArcS. Kinetic parameters of ArcS for lysine and tRNA-preQ0 were determined using purified enzyme. RNA fragments containing preQ0 were prepared from Saccharomyces cerevisiae tRNAPhe-preQ015. ArcS transferred 14C-labeled lysine to RNA fragments. Furthermore, ArcS transferred lysine to preQ0 nucleoside and preQ0 nucleoside 5'-monophosphate. Thus, the L-shaped structure and the sequence of tRNA are not essential for the lysine-transfer reaction by ArcS. However, the presence of D-arm structure accelerates the lysine-transfer reaction. Because ArcTGT from thermophilic archaea recognizes the common D-arm structure, we expected the combination of T. kodakarensis ArcTGT and ArcS and RaSEA complex would result in the formation of preQ0-Lys15 in all tRNAs. This hypothesis was confirmed using 46 T. kodakarensis tRNA transcripts and three H. volcanii tRNA transcripts. In addition, ArcTGT did not exchange the preQ0-Lys15 in tRNA with guanine or preQ0 base, showing that formation of tRNA-preQ0-Lys by ArcS plays a role in preventing the reverse reaction in G+ biosynthesis.

Identification of a radical SAM enzyme involved in the synthesis of archaeosine.

Archaeosine (G+), 7-formamidino-7-deazaguanosine, is an archaea-specific modified nucleoside found at the 15th position of tRNAs. In Euryarchaeota, 7-cyano-7-deazaguanine (preQ0)-containing tRNA (q0N-tRNA), synthesized by archaeal tRNA-guanine transglycosylase (ArcTGT), has been believed to be converted to G+-containing tRNA (G+-tRNA) by the paralog of ArcTGT, ArcS. However, we found that several euryarchaeal ArcSs have lysine transfer activity to q0N-tRNA to form q0kN-tRNA, which has a preQ0 lysine adduct as a base. Through comparative genomics and biochemical experiments, we found that ArcS forms a robust complex with a radical S-adenosylmethionine (SAM) enzyme named RaSEA. The ArcS-RaSEA complex anaerobically converted q0N-tRNA to G+-tRNA in the presence of SAM and lysine via q0kN-tRNA. We propose that ArcS and RaSEA should be considered an archaeosine synthase α-subunit (lysine transferase) and ß-subunit (q0kN-tRNA lyase), respectively.

Transcriptome Analysis and Identification of a Transcriptional Regulatory Network in the Response to H2O2.

Hydrogen peroxide (H2O2) is a common signal molecule initiating transcriptional responses to all the known biotic and abiotic stresses of land plants. However, the degree of involvement of H2O2 in these stress responses has not yet been well studied. Here we identify time-dependent transcriptome profiles stimulated by H2O2 application in Arabidopsis (Arabidopsis thaliana) seedlings. Promoter prediction based on transcriptome data suggests strong crosstalk among high light, heat, and wounding stress responses in terms of environmental stresses and between the abscisic acid (ABA) and salicylic acid (SA) responses in terms of phytohormone signaling. Quantitative analysis revealed that ABA accumulation is induced by H2O2 but SA is not, suggesting that the implied crosstalk with ABA is achieved through ABA accumulation while the crosstalk with SA is different. We identified potential direct regulatory pairs between regulator transcription factor (TF) proteins and their regulated TF genes based on the time-course transcriptome analysis for the H2O2 response, in vivo regulation of the regulated TF by the regulator TF identified by expression analysis of mutants and overexpressors, and in vitro binding of the regulator TF protein to the target TF promoter. These analyses enabled the establishment of part of the transcriptional regulatory network for the H2O2 response composed of 15 regulatory pairs of TFs, including five pairs previously reported. This regulatory network is suggested to be involved in a wide range of biotic and abiotic stress responses in Arabidopsis.

Multisite-specific archaeosine tRNA-guanine transglycosylase (ArcTGT) from Thermoplasma acidophilum, a thermo-acidophilic archaeon.

Archaeosine (G(+)), which is found only at position 15 in many archaeal tRNA, is formed by two steps, the replacement of the guanine base with preQ0 by archaeosine tRNA-guanine transglycosylase (ArcTGT) and the subsequent modification of preQ0 to G(+) by archaeosine synthase. However, tRNA(Leu) from Thermoplasma acidophilum, a thermo-acidophilic archaeon, exceptionally has two G(+)13 and G(+)15 modifications. In this study, we focused on the biosynthesis mechanism of G(+)13 and G(+)15 modifications in this tRNA(Leu). Purified ArcTGT from Pyrococcus horikoshii, for which the tRNA recognition mechanism and structure were previously characterized, exchanged only the G15 base in a tRNA(Leu) transcript with (14)C-guanine. In contrast, T. acidophilum cell extract exchanged both G13 and G15 bases. Because T. acidophilum ArcTGT could not be expressed as a soluble protein in Escherichia coli, we employed an expression system using another thermophilic archaeon, Thermococcus kodakarensis. The arcTGT gene in T. kodakarensis was disrupted, complemented with the T. acidophilum arcTGT gene, and tRNA(Leu) variants were expressed. Mass spectrometry analysis of purified tRNA(Leu) variants revealed the modifications of G(+)13 and G(+)15 in the wild-type tRNA(Leu). Thus, T. acidophilum ArcTGT has a multisite specificity and is responsible for the formation of both G(+)13 and G(+)15 modifications.

Correlation between the stability of tRNA tertiary structure and the catalytic efficiency of a tRNA-modifying enzyme, archaeal tRNA-guanine transglycosylase.

In many archaeal tRNAs, archaeosine is found at position 15. During archaeosine biosynthesis, archaeal tRNA-guanine transglycosylase (ArcTGT) first replaces the guanine base at position 15 with 7-cyano-7-deazaguanine (preQ0). In this study, we investigated whether modified nucleosides in tRNA substrates would affect ArcTGT incorporation of preQ0. We prepared a series of hypomodified tRNAs(Ser)(GGA) from Escherichia coli strains lacking each tRNA-modifying enzyme. Measurement of ArcTGT kinetic parameters with the various tRNAs(Ser)(GGA) as substrates showed that the Km decreased due to the lack of modified nucleosides. The tRNAs(Ser)(GGA) melting profiles resulted in experimental evidence showing that each modified nucleoside in tRNA(Ser)(GGA) enhanced tRNA stability. Furthermore, the ArcTGT K(m) strongly correlated with the melting temperature (T(m)), suggesting that the unstable tRNA containing fewer modified nucleosides served as a better ArcTGT substrate. These results show that preQ0 incorporation into tRNA by ArcTGT takes place early in the archaeal tRNA modification process.

Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate.

BACKGROUND: Angiotensinogen (ANG) is a macromolecular precursor of angiotensin, which regulates blood pressure and electrolyte balance. ANG is specifically cleaved by renin, an aspartic protease, to initiate the angiotensin-processing cascade. Ovine ANG (oANG) from sheep plasma has been shown to be a better substrate for human renin, and it has been used in clinical renin assays. To expand the availability of oANG, we aimed to produce milligram levels of recombinant oANG using an Escherichia coli expression system. RESULTS: When recombinant oANG was expressed from a T7 promoter in various E. coli strains at 37 °C, it accumulated in the insoluble fraction. However, by expressing oANG at 37 °C from a tac promoter, which has weaker transcriptional activity than a T7 promoter, we significantly elevated the ratio of soluble to insoluble recombinant oANG. Using a novel culturing system and auto-induction culture medium, we purified tac-expressed recombinant oANG to homogeneity, with a yield of 4.0 mg per liter of culture. Based on size-exclusion gel filtration analysis and dynamic light scattering analysis, the resulting purified oANG is a monomer in solution. The circular dichroism spectrum of E. coli-expressed recombinant oANG was similar to that of oANG expressed in CHO cells. Differential scanning fluorimetry showed that both preparations undergo a two-state transition during thermal denaturation, and the melting temperatures of recombinant oANG expressed in E. coli and CHO cells were 49.4 ± 0.16 °C and 51.6 ± 0.19 °C, respectively. The K(m) values of both oANG preparations were similar; the k(cat) value of E. coli-expressed recombinant oANG was slightly higher than that of CHO-expressed oANG. CONCLUSIONS: Recombinant oANG expressed in E. coli functions as a human renin substrate. This study presents an E. coli-based system for the rapid production of milligram quantities of a human renin substrate, which will be useful for both fundamental and clinical studies on renin and hypertension.

Involvement of plasmin-mediated extracellular activation of progalanin in angiogenesis.

Progalanin is released from the small cell lung carcinoma line SBC-3A and converted to its active form by plasmin. To elucidate the role of progalanin activation in the extracellular compartment, matrix metalloproteinase (MMP) activity was studied in SBC-3A cells treated with progalanin siRNA, and angiogenesis was measured in tumor tissue originating from SBC-3A cell transplantation into mice. Progalanin siRNA caused downregulation of progalanin expression for approximately 8 days. MMP activity and angiogenesis were reduced in tumors induced by transplantation of progalanin siRNA-treated SBC-3A cells. In contrast, MMP-9 and MMP-2 activity and angiogenesis increased in tumors originating from progalanin siRNA-treated SBC-3A cells in the presence of galanin and progalanin. Furthermore, injection of tranexamic acid, a plasmin inhibitor, more markedly reduced MMP-9 and MMP-2 activity and angiogenesis in tumors originating from progalanin siRNA-treated SBC-3A cells and in tumor tissue originating from progalanin siRNA-treated SBC-3A cells in the presence of progalanin. The reduction of MMP-9 and MMP-2 activity with tranexamic acid was restored by galanin, but not by progalanin. Moreover, tranexamic acid reduced angiogenesis in control siRNA-treated SBC-3A cells. These results suggest that the activation of progalanin by plasmin in the extracellular compartment was involved in MMP-9 and MMP-2 activation and in angiogenesis in tumor tissue.

Purification and comparison of native and recombinant tRNA-guanine transglycosylases from Methanosarcina acetivorans.

Many archaeal tRNAs have archaeosine (G(+)) at position 15 in the D-loop and this is thought to strengthen the tertiary interaction with C48 in the V-loop. In the first step of G(+) biosynthesis, archaeosine tRNA-guanine transglycosylase (ArcTGT)(1) catalyzes the base exchange reaction from guanine to 7-cyano-7-deazaguanine (preQ(0)). ArcTGT is classified into full-size or split types, according to databases of genomic information. Although the full-size type forms a homodimeric structure, the split type has been assumed to form a heterotetrameric structure, consisting of two kinds of peptide. However, there has been no definitive evidence for this presented to date. Here, we show that native ArcTGT could be isolated from Methanosarcina acetivorans and two peptides formed a robust complex in cells. Consequently, the two peptides function as actual subunits of ArcTGT. We also overexpressed recombinant ArcTGT in Escherichia coli cells. Product was successfully obtained by co-overexpression of the two subunits but one subunit alone was not adequately expressed in soluble fractions. This result suggests that interaction between the two subunits may contribute to the conformational stability of split ArcTGT. The values of the kinetic parameters for the recombinant and native ArcTGT were closely similar. Moreover, tRNA transcript with preQ(0) at position 15 was successfully prepared using the recombinant ArcTGT. This tRNA transcript is expected to be useful as a substrate for studies seeking the enzymes responsible for G(+) biosynthesis.

Corrigendum to "establishing a novel assay system for measuring renin concentration using cost effective recombinant ovine angiotensinogen".

[This corrects the article DOI: 10.1016/j.heliyon.2019.e01409.].

Site-specific attachment of a protein to a carbon nanotube end without loss of protein function.

Establishing a nanobiohybrid device largely relies on the availability of various bioconjugation procedures which allow coupling of biomolecules and inorganic materials. Especially, site-specific coupling of a protein to nanomaterials is highly useful and significant, since it can avoid adversely affecting the protein's function. In this study, we demonstrated a covalent coupling of a protein of interest to the end of carbon nanotubes without affecting protein's function. A modified Staudinger-Bertozzi ligation was utilized to couple a carbon nanotube end with an azide group which is site-specifically incorporated into a protein of interest. We demonstrated that Ca(2+)-sensor protein, calmodulin, can be attached to the end of the nanotubes without affecting the ability to bind to the substrate in a calcium-dependent manner. This procedure can be applied not only to nanotubes, but also to other nanomaterials, and therefore provides a fundamental technique for well-controlled protein conjugation.

Agmatine-conjugated cytidine in a tRNA anticodon is essential for AUA decoding in archaea.

A modified base at the first (wobble) position of some tRNA anticodons is critical for deciphering the genetic code. In eukaryotes and eubacteria, AUA codons are decoded by tRNAsIle with modified bases pseudouridine (and/or inosine) and lysidine, respectively. The mechanism by which archaeal species translate AUA codons is unclear. We describe a polyamine-conjugated modified base, 2-agmatinylcytidine (agm(2)C or agmatidine), at the wobble position of archaeal tRNA(Ile) that decodes AUA codons specifically. We demonstrate that archaeal cells use agmatine to synthesize agm(2)C of tRNA(Ile). We also identified a new enzyme, tRNA(Ile)-agm(2)C synthetase (TiaS), that catalyzes agm(2)C formation in the presence of agmatine and ATP. Although agm(2)C is chemically similar to lysidine, TiaS constitutes a distinct class of enzyme from tRNA(Ile)-lysidine synthetase (TilS), suggesting that the decoding systems evolved convergently across domains.

N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network.

N(7)-methylguanine at position 46 (m(7)G46) in tRNA is produced by tRNA (m(7)G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (DeltatrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the DeltatrmB strain and the lack of the m(7)G46 modification in tRNA(Phe) were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the DeltatrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m(7)G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m(1)G37, suggesting that the m(7)G46 positively affects their formations. Although the lack of the m(7)G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA(Phe), they cause a decrease in melting temperature of class I tRNA and degradation of tRNA(Phe) and tRNA(Ile). (35)S-Met incorporation into proteins revealed that protein synthesis in DeltatrmB cells is depressed above 70 degrees C. At 80 degrees C, the DeltatrmB strain exhibits a severe growth defect. Thus, the m(7)G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m(7)G46 modification supports introduction of other modifications.

Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts.

We found that both tetramethylammonium chloride (TMA-Cl) and tetra-ethylammonium chloride (TEA-Cl), which are used as monovalent cations for northern hybridization, drastically destabilized the tertiary structures of tRNAs and enhanced the formation of tRNA*oligoDNA hybrids. These effects are of great advantage for the hybridization-based method for purification of specific tRNAs from unfractionated tRNA mixtures through the use of an immobilized oligoDNA complementary to the target tRNA. Replacement of NaCl by TMA-Cl or TEA-Cl in the hybridization buffer greatly improved the recovery of a specific tRNA, even from unfractionated tRNAs derived from a thermophile. Since TEA-Cl destabilized tRNAs more strongly than TMA-Cl, it was necessary to lower the hybridization temperature at the sacrifice of the purity of the recovered tRNA when using TEA-Cl. Therefore, we propose two alternative protocols, depending on the desired properties of the tRNA to be purified. When the total recovery of the tRNA is important, hybridization should be carried out in the presence of TEA-Cl. However, if the purity of the recovered tRNA is important, TMA-Cl should be used for the hybridization. In principle, this procedure for tRNA purification should be applicable to any small-size RNA whose gene sequence is already known.

Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion.

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase-tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS-tRNA(Tyr) pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA(Tyr). The endogenous TyrRS and tRNA(Tyr) genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS-tRNA(Tyr) pair. In this engineered strain, 3-iodo-L-tyrosine and 3-azido-L-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-L-tyrosine and was also found to recognize 3-azido-L-tyrosine. The structural basis for the 3-azido-L-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.

Unconventional decoding of the AUA codon as methionine by mitochondrial tRNAMet with the anticodon f5CAU as revealed with a mitochondrial in vitro translation system.

Mitochondrial (mt) tRNA(Met) has the unusual modified nucleotide 5-formylcytidine (f(5)C) in the first position of the anticodon. This tRNA must translate both AUG and AUA as methionine. By constructing an in vitro translation system from bovine liver mitochondria, we examined the decoding properties of the native mt tRNA(Met) carrying f(5)C in the anticodon compared to a transcript that lacks the modification. The native mt Met-tRNA could recognize both AUA and AUG codons as Met, but the corresponding synthetic tRNA(Met) lacking f(5)C (anticodon CAU), recognized only the AUG codon in both the codon-dependent ribosomal binding and in vitro translation assays. Furthermore, the Escherichia coli elongator tRNA(Met)(m) with the anticodon ac(4)CAU (ac(4)C = 4-acetylcytidine) and the bovine cytoplasmic initiator tRNA(Met) (anticodon CAU) translated only the AUG codon for Met on mt ribosome. The codon recognition patterns of these tRNAs were the same on E. coli ribosomes. These results demonstrate that the f(5)C modification in mt tRNA(Met) plays a crucial role in decoding the nonuniversal AUA codon as Met, and that the genetic code variation is compensated by a change in the tRNA anticodon, not by a change in the ribosome. Base pairing models of f(5)C-G and f(5)C-A based on the chemical properties of f(5)C are presented.

Boosting Auto-Induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives.

BACKGROUND: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. OBJECTIVES: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. METHODS: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression). RESULTS: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. CONCLUSION: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

Aquifex aeolicus tRNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes transfer of methyl groups not only to guanine 26 but also to guanine 27 in tRNA.

Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m(2)(2)G26) in tRNA. In the reaction, N2-guanine at position 26 (m(2)G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA(Cys) has an m(2)(2)G26m(2)G27 or m(2)(2)G26m(2)(2)G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m(2)G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

Application of solid-phase DNA probe method with cleavage by deoxyribozyme for analysis of long non-coding RNAs.

The solid-phase DNA probe method is a well-established technique for tRNA purification. We have applied this method for purification and analysis of other non-coding RNAs. Three columns for purification of tRNAPhe, transfer-messenger RNA (tmRNA) and 16S rRNA from Thermus thermophilus were connected in tandem and purifications were performed. From each column, tRNAPhe, tmRNA and 16S rRNA could be purified in a single step. This is the first report of purification of native tmRNA from T. thermophilus and the purification demonstrates that the solid-phase DNA probe method is applicable to non-coding RNA, which is present in lower amounts than tRNA. Furthermore, if a long non-coding RNA is cleaved site-specifically and the fragment can be purified by the solid-phase DNA probe method, modified nucleosides in the long non-coding RNA can be analysed. Therefore, we designed a deoxyribozyme (DNAzyme) to perform site-specific cleavage of 16S rRNA, examined optimum conditions and purified the resulting RNA fragment. Sequencing of complimentary DNA and mass spectrometric analysis revealed that the purified RNA corresponded to the targeted fragment of 16S rRNA. Thus, the combination of DNAzyme cleavage and purification using solid-phase DNA probe methodology can be a useful technique for analysis of modified nucleosides in long non-coding RNAs.

Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs.

Entire reconstitution of tRNAs for active protein production in a cell-free system brings flexibility into the genetic code engineering. It can also contribute to the field of cell-free synthetic biology, which aims to construct self-replicable artificial cells. Herein, we developed a system equipped only with in vitro transcribed tRNA (iVTtRNA) based on a reconstituted cell-free protein synthesis (PURE) system. The developed system, consisting of 21 iVTtRNAs without nucleotide modifications, is able to synthesize active proteins according to the redesigned genetic code. Manipulation of iVTtRNA composition in the system enabled genetic code rewriting. Introduction of modified nucleotides into specific iVTtRNAs demonstrated to be effective for both protein yield and decoding fidelity, where the production yield of DHFR reached about 40% of the reaction with native tRNA at 30°C. The developed system will prove useful for studying decoding processes, and may be employed in genetic code and protein engineering applications.