The Experts below are selected from a list of 4653 Experts worldwide ranked by ideXlab platform
Hiroaki Suga - One of the best experts on this subject based on the ideXlab platform.
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an Aminoacylation ribozyme evolved from a natural tRNA sensing t box riboswitch
Nature Chemical Biology, 2020Co-Authors: Satoshi Ishida, Naohiro Terasaka, Takayuki Katoh, Hiroaki SugaAbstract:When the primitive translation system first emerged in the hypothetical RNA world, ribozymes could have been responsible for Aminoacylation. Given that naturally occurring T-box riboswitches selectively sense the Aminoacylation status of cognate tRNAs, we introduced a domain of random sequence into a T-box–tRNA conjugate and isolated ribozymes that were self-aminoacylating on the 3′-terminal hydroxyl group. One of them, named Tx2.1, recognizes the anticodon and D-loop of tRNA via interaction with its stem I domain, similarly to the parental T-box, and selectively charges N-biotinyl-l-phenylalanine (Bio-lPhe) onto the 3′ end of the cognate tRNA in trans. We also demonstrated the ribosomal synthesis of a Bio-lPhe-initiated peptide in a Tx2.1-coupled in vitro translation system, in which Tx2.1 catalyzed specific tRNA Aminoacylation in situ. This suggests that such ribozymes could have coevolved with a primitive translation system in the RNA world. The authors develop a ribozyme, Tx2.1, that is capable of aminoacylating tRNA with specificity for the anticodon from directed evolution of a T-box riboswitch. Tx2.1 could be used to charge non-natural amino acids in an in vitro translation system.
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the rna origin of transfer rna Aminoacylation and beyond
Philosophical Transactions of the Royal Society B, 2011Co-Authors: Hiroaki Suga, Gosuke Hayashi, Naohiro TerasakaAbstract:Aminoacylation of tRNA is an essential event in the translation system. Although in the modern system protein enzymes play the sole role in tRNA Aminoacylation, in the primitive translation system RNA molecules could have catalysed Aminoacylation onto tRNA or tRNA-like molecules. Even though such RNA enzymes so far are not identified from known organisms, in vitro selection has generated such RNA catalysts from a pool of random RNA sequences. Among them, a set of RNA sequences, referred to as flexizymes (Fxs), discovered in our laboratory are able to charge amino acids onto tRNAs. Significantly, Fxs allow us to charge a wide variety of amino acids, including those that are non-proteinogenic, onto tRNAs bearing any desired anticodons, and thus enable us to reprogramme the genetic code at our will. This article summarizes the evolutionary history of Fxs and also the most recent advances in manipulating a translation system by integration with Fxs.
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structural basis of specific tRNA Aminoacylation by a small in vitro selected ribozyme
Nature, 2008Co-Authors: Hong Xiao, Hiroaki Suga, Hiroshi Murakami, Adrian R FerredamareAbstract:In modern organisms, protein enzymes are solely responsible for the Aminoacylation of transfer RNA. However, the evolution of protein synthesis in the RNA world required RNAs capable of catalysing this reaction. Ribozymes that aminoacylate RNA by using activated amino acids have been discovered through selection in vitro. Flexizyme is a 45-nucleotide ribozyme capable of charging tRNA in trans with various activated l-phenylalanine derivatives. In addition to a more than 10(5) rate enhancement and more than 10(4)-fold discrimination against some non-cognate amino acids, this ribozyme achieves good regioselectivity: of all the hydroxyl groups of a tRNA, it exclusively aminoacylates the terminal 3'-OH. Here we report the 2.8-A resolution structure of flexizyme fused to a substrate RNA. Together with randomization of ribozyme core residues and reselection, this structure shows that very few nucleotides are needed for the Aminoacylation of specific tRNAs. Although it primarily recognizes tRNA through base-pairing with the CCA terminus of the tRNA molecule, flexizyme makes numerous local interactions to position the acceptor end of tRNA precisely. A comparison of two crystallographically independent flexizyme conformations, only one of which appears capable of binding activated phenylalanine, suggests that this ribozyme may achieve enhanced specificity by coupling active-site folding to tRNA docking. Such a mechanism would be reminiscent of the mutually induced fit of tRNA and protein employed by some aminoacyl-tRNA synthetases to increase specificity.
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the flexizyme system a highly flexible tRNA Aminoacylation tool for the translation apparatus
Current Opinion in Chemical Biology, 2007Co-Authors: Masaki Ohuchi, Hiroshi Murakami, Hiroaki SugaAbstract:Flexizymes are de novo ribozymes capable of charging a wide variety of non-natural amino acids on tRNAs. The flexizyme system enables reprogramming of the genetic code by reassigning the codons that are generally assigned to natural amino acids to non-natural residues, and thus mRNA-directed synthesis of non-natural polypeptides can be achieved. In this review, we comprehensively summarize the history of the flexizyme system and its subsequent development into a practical tool. Furthermore, applications to the synthesis of novel biopolymers via genetic code reprogramming and perspectives for future applications are described.
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using a solid phase ribozyme Aminoacylation system to reprogram the genetic code
Chemistry & Biology, 2003Co-Authors: Hiroshi Murakami, Dimitrios Kourouklis, Hiroaki SugaAbstract:Here, we report a simple and economical tRNA Aminoacylation system based upon a resin-immobilized ribozyme, referred to as Flexiresin. This catalytic system features a broad spectrum of activities toward various phenylalanine (Phe) analogs and suppressor tRNAs. Most importantly, it allows users to perform the tRNA Aminoacylation reaction and isolate the aminoacylated tRNAs in a few hours. We coupled the Flexiresin system with a high-performance cell-free translation system and demonstrated protein mutagenesis with seven different Phe analogs in parallel. Thus, the technology developed herein provides a new tool that significantly simplifies the procedures for the synthesis of aminoacyl-tRNAs charged with nonnatural amino acids, which makes the nonnatural amino acid mutagenesis of proteins more user accessible.
Enduo Wang - One of the best experts on this subject based on the ideXlab platform.
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hearing impairment associated kars mutations lead to defects in Aminoacylation of both cytoplasmic and mitochondrial tRNA lys
Science China-life Sciences, 2020Co-Authors: Enduo Wang, Yong Wang, Meiqin Xue, Jingbo Zhou, Qiyu Zeng, Pengfei Fang, Xiaolong ZhouAbstract:Aminoacyl-tRNA synthetases (aaRSs) are ubiquitously expressed, essential enzymes, synthesizing aminoacyl-tRNAs for protein synthesis. Functional defects of aaRSs frequently cause various human disorders. Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases (LysRSs). Previously, two mutations (c.1129G>A and c.517T>C) were identified that led to hearing impairment; however, the underlying biochemical mechanism is unclear. In the present study, we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol, but affect the cytosolic LysRS level, its tertiary structure, and cytosolic tRNA Aminoacylation in vitro. As for mitochondrial translation, the two mutations have little effect on the steady-state level, mitochondrial targeting, and tRNA binding affinity of mitochondrial LysRS. However, they exhibit striking differences in charging mitochondrial tRNALys, with the c.517T>C mutant being completely deficient in vitro and in vivo. We constructed two yeast genetic models, which are powerful tools to test the in vivo Aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels. Overall, our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.
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a threonyl tRNA synthetase like protein has tRNA Aminoacylation and editing activities
Nucleic Acids Research, 2018Co-Authors: Yun Chen, Xiaolong Zhou, Enduo Wang, Zhirong Ruan, Yong Wang, Qian Huang, Meiqin XueAbstract:TARS and TARS2 encode cytoplasmic and mitochondrial threonyl-tRNA synthetases (ThrRSs) in mammals, respectively. Interestingly, in higher eukaryotes, a third gene, TARSL2, encodes a ThrRS-like protein (ThrRS-L), which is highly homologous to cytoplasmic ThrRS but with a different N-terminal extension (N-extension). Whether ThrRS-L has canonical functions is unknown. In this work, we studied the organ expression pattern, cellular localization, canonical Aminoacylation and editing activities of mouse ThrRS-L (mThrRS-L). Tarsl2 is ubiquitously but unevenly expressed in mouse tissues. Different from mouse cytoplasmic ThrRS (mThrRS), mThrRS-L is located in both the cytoplasm and nucleus; the nuclear distribution is mediated via a nuclear localization sequence at its C-terminus. Native mThrRS-L enriched from HEK293T cells was active in Aminoacylation and editing. To investigate the in vitro catalytic properties of mThrRS-L accurately, we replaced the N-extension of mThrRS-L with that of mThrRS. The chimeric protein (mThrRS-L-NT) has amino acid activation, Aminoacylation and editing activities. We compared the activities and cross-species tRNA recognition between mThrRS-L-NT and mThrRS. Despite having a similar Aminoacylation activity, mThrRS-L-NT and mThrRS exhibit differences in tRNA recognition and editing capacity. Our results provided the first analysis of the Aminoacylation and editing activities of ThrRS-L, and improved our understanding of Tarsl2.
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Aminoacylation and translational quality control strategy employed by leucyl tRNA synthetase from a human pathogen with genetic code ambiguity
Nucleic Acids Research, 2013Co-Authors: Xiaolong Zhou, Zhirong Ruan, Zhipeng Fang, Meng Wang, Rujuan Liu, Min Tan, Fabrizio Anella, Enduo WangAbstract:Aminoacyl-tRNA synthetases should ensure high accuracy in tRNA Aminoacylation. However, the absence of significant structural differences between amino acids always poses a direct challenge for some aminoacyl-tRNA synthetases, such as leucyl-tRNA synthetase (LeuRS), which require editing function to remove mis-activated amino acids. In the cytoplasm of the human pathogen Candida albicans, the CUG codon is translated as both Ser and Leu by a uniquely evolved CatRNA(Ser)(CAG). Its cytoplasmic LeuRS (CaLeuRS) is a crucial component for CUG codon ambiguity and harbors only one CUG codon at position 919. Comparison of the activity of CaLeuRS-Ser(919) and CaLeuRS-Leu(919) revealed yeast LeuRSs have a relaxed tRNA recognition capacity. We also studied the mis-activation and editing of non-cognate amino acids by CaLeuRS. Interestingly, we found that CaLeuRS is naturally deficient in tRNA-dependent pre-transfer editing for non-cognate norvaline while displaying a weak tRNA-dependent pre-transfer editing capacity for non-cognate α-amino butyric acid. We also demonstrated that post-transfer editing of CaLeuRS is not tRNA(Leu) species-specific. In addition, other eukaryotic but not archaeal or bacterial LeuRSs were found to recognize CatRNA(Ser)(CAG). Overall, we systematically studied the Aminoacylation and editing properties of CaLeuRS and established a characteristic LeuRS model with naturally deficient tRNA-dependent pre-transfer editing, which increases LeuRS types with unique editing patterns.
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the yin and yang of tRNA proper binding of acceptor end determines the catalytic balance of editing and Aminoacylation
Nucleic Acids Research, 2013Co-Authors: Min Tan, Xiaolong Zhou, Meng Wang, Wei Yan, Gilbert Eriani, Enduo WangAbstract:Faithful translation of the genetic code depends on accurate coupling of amino acids with cognate transfer RNAs (tRNAs) catalyzed by aminoacyl-tRNA synthetases. The fidelity of leucyl-tRNA synthetase (LeuRS) depends mainly on proofreading at the pre- and post-transfer levels. During the catalytic cycle, the tRNA CCA-tail shuttles between the synthetic and editing domains to accomplish the Aminoacylation and editing reactions. Previously, we showed that the Y330D mutation of Escherichia coli LeuRS, which blocks the entry of the tRNA CCA-tail into the connective polypeptide 1domain, abolishes both tRNA-dependent pre- and post-transfer editing. In this study, we identified the counterpart substitutions, which constrain the tRNA acceptor stem binding within the synthetic active site. These mutations negatively impact the tRNA charging activity while retaining the capacity to activate the amino acid. Interestingly, the mutated LeuRSs exhibit increased global editing activity in the presence of a non-cognate amino acid. We used a reaction mimicking post-transfer editing to show that these mutations decrease post-transfer editing owing to reduced tRNA Aminoacylation activity. This implied that the increased editing activity originates from tRNA-dependent pre-transfer editing. These results, together with our previous work, provide a comprehensive assessment of how intra-molecular translocation of the tRNA CCA-tail balances the Aminoacylation and editing activities of LeuRS.
Elena L Paley - One of the best experts on this subject based on the ideXlab platform.
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Paley_Manuscript_Supplementary__3-22-2019 – Supplemental material for Diet-Related Metabolic Perturbations of Gut Microbial Shikimate Pathway-Tryptamine-tRNA Aminoacylation-Protein Synthesis in Human Health and Disease
2019Co-Authors: Elena L PaleyAbstract:Supplemental material, Paley_Manuscript_Supplementary__3-22-2019 for Diet-Related Metabolic Perturbations of Gut Microbial Shikimate Pathway-Tryptamine-tRNA Aminoacylation-Protein Synthesis in Human Health and Disease by Elena L Paley in International Journal of Tryptophan Research
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Diet-Related Metabolic Perturbations of Gut Microbial Shikimate Pathway-Tryptamine-tRNA Aminoacylation-Protein Synthesis in Human Health and Disease
SAGE Publishing, 2019Co-Authors: Elena L PaleyAbstract:Human gut bacterial Na(+)-transporting NADH:ubiquinone reductase (NQR) sequence is associated with Alzheimer disease (AD). Here, Alzheimer disease-associated sequence (ADAS) is further characterized in cultured spore-forming Clostridium sp . Tryptophan and NQR substrate ubiquinone have common precursor chorismate in microbial shikimate pathway. Tryptophan-derived tryptamine presents in human diet and gut microbiome. Tryptamine inhibits tryptophanyl-tRNA synthetase (TrpRS) with consequent neurodegeneration in cell and animal models. Tryptophanyl-tRNA synthetase inhibition causes protein biosynthesis impairment similar to that revealed in AD. Tryptamine-induced TrpRS gene-dose reduction is associated with TrpRS protein deficiency and cell death. In animals, tryptamine treatment results in toxicity, weight gain, and prediabetes-related hypoglycemia. Sequence analysis of gut microbiome database reveals 89% to 100% ADAS nucleotide identity in American Indian (Cheyenne and Arapaho [C&A]) Oklahomans, of which ~93% being overweight or obese and 50% self-reporting type 2 diabetes (T2D). Alzheimer disease-associated sequence occurs in 10.8% of C&A vs 1.3% of healthy American population. This observation is of considerable interest because T2D links to AD and obesity. Alzheimer disease-associated sequence prevails in gut microbiome of colorectal cancer, which linked to AD. Metabolomics revealed that tryptamine, chorismate precursor quinate, and chorismate product 4-hydroxybenzoate (ubiquinone precursor) are significantly higher, while tryptophan-containing dipeptides are lower due to tRNA Aminoacylation deficiency in C&A compared with non-native Oklahoman who showed no ADAS. Thus, gut microbial tryptamine overproduction correlates with ADAS occurrence. Antibiotic and diet additives induce ADAS and tryptamine. Mitogenic/cytotoxic tryptamine cause microbial and human cell death, gut dysbiosis, and consequent disruption of host-microbe homeostasis. Present analysis of 1246 participants from 17 human gut metagenomics studies revealed ADAS in cell death diseases
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towards an integrative understanding of tRNA Aminoacylation diet host gut microbiome interactions in neurodegeneration
Nutrients, 2018Co-Authors: Elena L Paley, George PerryAbstract:Transgenic mice used for Alzheimer's disease (AD) preclinical experiments do not recapitulate the human disease. In our models, the dietary tryptophan metabolite tryptamine produced by human gut microbiome induces tryptophanyl-tRNA synthetase (TrpRS) deficiency with consequent neurodegeneration in cells and mice. Dietary supplements, antibiotics and certain drugs increase tryptamine content in vivo. TrpRS catalyzes tryptophan attachment to tRNAtrp at initial step of protein biosynthesis. Tryptamine that easily crosses the blood-brain barrier induces vasculopathies, neurodegeneration and cell death via TrpRS competitive inhibition. TrpRS inhibitor tryptophanol produced by gut microbiome also induces neurodegeneration. TrpRS inhibition by tryptamine and its metabolites preventing tryptophan incorporation into proteins lead to protein biosynthesis impairment. Tryptophan, a least amino acid in food and proteins that cannot be synthesized by humans competes with frequent amino acids for the transport from blood to brain. Tryptophan is a vulnerable amino acid, which can be easily lost to protein biosynthesis. Some proteins marking neurodegenerative pathology, such as tau lack tryptophan. TrpRS exists in cytoplasmic (WARS) and mitochondrial (WARS2) forms. Pathogenic gene variants of both forms cause TrpRS deficiency with consequent intellectual and motor disabilities in humans. The diminished tryptophan-dependent protein biosynthesis in AD patients is a proof of our model-based disease concept.
Susan A. Martinis - One of the best experts on this subject based on the ideXlab platform.
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tRNA synthetase tRNA Aminoacylation and beyond
Wiley Interdisciplinary Reviews - Rna, 2014Co-Authors: Yan Ling Joy Pang, Kiranmai Poruri, Susan A. MartinisAbstract:The aminoacyl-tRNA synthetases are prominently known for their classic function in the first step of protein synthesis, where they bear the responsibility of setting the genetic code. Each enzyme is exquisitely adapted to covalently link a single standard amino acid to its cognate set of tRNA isoacceptors. These ancient enzymes have evolved idiosyncratically to host alternate activities that go far beyond their Aminoacylation role and impact a wide range of other metabolic pathways and cell signaling processes. The family of aminoacyl-tRNA synthetases has also been suggested as a remarkable scaffold to incorporate new domains that would drive evolution and the emergence of new organisms with more complex function. Because they are essential, the tRNA synthetases have served as pharmaceutical targets for drug and antibiotic development. The recent unfolding of novel important functions for this family of proteins offers new and promising pathways for therapeutic development to treat diverse human diseases.
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a unique insert of leucyl tRNA synthetase is required for Aminoacylation and not amino acid editing
Biochemistry, 2007Co-Authors: Susan A. MartinisAbstract:Leucyl-tRNA synthetase (LeuRS) is a class I enzyme, which houses its Aminoacylation active site in a canonical core that is defined by a Rossmann nucleotide binding fold. In addition, many LeuRSs bear a unique polypeptide insert comprised of about 50 amino acids located just upstream of the conserved KMSKS sequence. The role of this leucine-specific domain (LS-domain) remains undefined. We hypothesized that this domain may be important for substrate recognition in Aminoacylation and/or amino acid editing. We carried out a series of deletion mutations and chimeric swaps within the leucine-specific domain of Escherichia coli. Our results support that the leucine-specific domain is critical for Aminoacylation but not required for editing activity. Kinetic analysis determined that deletion of the LS-domain primarily impacts k cat . Because of its proximity to the Aminoacylation active site, we propose that this domain interacts with the tRNA during amino acid activation and/or tRNA Aminoacylation. Although the leucine-specific domain does not appear to be important to the editing complex, it remains possible that it aids the dynamic translocation process that moves tRNA from the Aminoacylation to the editing complex.
Daniel Kern - One of the best experts on this subject based on the ideXlab platform.
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The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis.
Molecular Cell, 2007Co-Authors: Marc Bailly, Mickael Blaise, Hubert Dominique Becker, Bernard Lorber, Daniel KernAbstract:Asparagine, one of the 22 genetically encoded amino acids, can be synthesized by a tRNA-dependent mechanism. So far, this type of pathway was believed to proceed via two independent steps. A nondiscriminating aspartyl-tRNA synthetase (ND-DRS) first generates a mischarged aspartyl-tRNAAsn that dissociates from the enzyme and binds to a tRNA-dependent amidotransferase (AdT), which then converts the tRNA-bound aspartate into asparagine. We show herein that the ND-DRS, tRNAAsn, and AdT assemble into a specific ribonucleoprotein complex called transamidosome that remains stable during the overall catalytic process. Our results indicate that the tRNAAsn-mediated linkage between the ND-DRS and AdT enables channeling of the mischarged aspartyl-tRNAAsn intermediate between DRS and AdT active sites to prevent challenging of the genetic code integrity. We propose that formation of a ribonucleoprotein is a general feature for tRNA-dependent amino acid biosynthetic pathways that are remnants of earlier stages when amino acid synthesis and tRNA Aminoacylation were coupled.
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non discriminating and discriminating aspartyl tRNA synthetases differ in the anticodon binding domain
The EMBO Journal, 2003Co-Authors: Christophe Charron, Mickael Blaise, Herve Roy, Richard Giege, Daniel KernAbstract:In most organisms, tRNA Aminoacylation is ensured by 20 aminoacyl-tRNA synthetases (aaRSs). In eubacteria, however, synthetases can be duplicated as in Thermus thermophilus, which contains two distinct AspRSs. While AspRS-1 is specific, AspRS-2 is non-discriminating and aspartylates tRNAAsp and tRNAAsn. The structure at 2.3 Å resolution of AspRS-2, the first of a non-discriminating synthetase, was solved. It differs from that of AspRS-1 but has resemblance to that of discriminating and archaeal AspRS from Pyrococcus kodakaraensis. The protein presents non-conventional features in its OB-fold anticodon-binding domain, namely the absence of a helix inserted between two β-strands of this fold and a peculiar L1 loop differing from the large loops known to interact with tRNAAsp identity determinant C36 in conventional AspRSs. In AspRS-2, this loop is small and structurally homologous to that in AsnRSs, including conservation of a proline. In discriminating Pyrococcus AspRS, the L1 loop, although small, lacks this proline and is not superimposable with that of AspRS-2 or AsnRS. Its particular status is demonstrated by a loop-exchange experiment that renders the Pyrococcus AspRS non-discriminating.
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thermus thermophilus a link in evolution of the tRNA dependent amino acid amidation pathways
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Hubert Dominique Becker, Daniel KernAbstract:Thermus thermophilus possesses an aspartyl-tRNA synthetase (AspRS2) able to aspartylate efficiently tRNAAsp and tRNAAsn. Aspartate mischarged on tRNAAsn then is converted into asparagine by an ω amidase that differs structurally from all known asparagine synthetases. However, aspartate is not misincorporated into proteins because the binding capacity of aminoacylated tRNAAsn to elongation factor Tu is only conferred by conversion of aspartate into asparagine. T. thermophilus additionally contains a second aspartyl-tRNA synthetase (AspRS1) able to aspartylate tRNAAsp and an asparaginyl-tRNA synthetase able to charge tRNAAsn with free asparagine, although the organism does not contain a tRNA-independent asparagine synthetase. In contrast to the duplicated pathway of tRNA asparaginylation, tRNA glutaminylation occurs in the thermophile via the usual pathway by using glutaminyl-tRNA synthetase and free glutamine synthesized by glutamine synthetase that is unique. T. thermophilus is able to ensure tRNA Aminoacylation by alternative routes involving either the direct pathway or by conversion of amino acid mischarged on tRNA. These findings shed light on the interrelation between the tRNA-dependent and tRNA-independent pathways of amino acid amidation and on the processes involved in fidelity of the Aminoacylation systems.
