Cognate tRNA Research Articles

Dual Pathways for B. anthracis Asparaginyl‐tRNA Formation

In Bacillus anthracis, there are two distinct routes for attaching asparagine (Asn) to its cognate transfer RNA (tRNAAsn), an essential step in protein synthesis. In the direct route, asparaginyl‐tRNA synthetase (AsnRS) directly ligates Asn to tRNAAsn. The indirect pathway is a two‐step process where a non‐discriminating‐AspRS (ND‐AspRS) attaches aspartate (Asp) to tRNAAsn which is them amidated by GatCAB, an amidotransferase. In this pathway, Asn is ultimately synthesized on the tRNA using a complex called the transamidosome which is made up of the ND‐AspRS, tRNAAsn, and GatCAB. B. anthracis encodes two distinct AspRSs with one of bacterial origins and the other acquired through horizontal gene transfer from archaea. The archaeal AspRS serves as the non‐discriminating enzyme for tRNA‐dependent asparagine biosynthesis. We have successfully overproduced in E. coli and purified the enzymes for both pathways to further characterize the two routes under various physiologically relevant conditions. The research provides a better understanding as to why this pathogen acquired an archaeal aspartyl‐tRNA synthetase and encodes two distinct pathways for asparaginyl‐tRNA synthesis.

Targeting GCN2 Regulation of Amino Acid Homeostasis in Prostate Cancer

The Integrated Stress Response (ISR) plays a critical role in the adaptation and survival of tumor cells to exogenous and endogenous stresses. The ISR features four protein kinases (PERK, GCN2, PKR, and HRI), each activated by different stresses, that phosphorylate the eukaryotic translation initiation factor eIF2, resulting in repression of global protein synthesis. Paradoxically, eIF2 phosphorylation also enhances translation of select gene transcripts, including the transcription factor ATF4, which is central for ISR‐directed gene transcription. Therefore, the ISR directs translational and transcriptional control that is critical for cancer stress adaptation. Moreover, eIF2 phosphorylation and ATF4 have recently been suggested to play a role in prostate cancer (PCa) growth and survival; however, the specific function of ISR kinases, their mode of activation, and the mechanisms by which the ISR facilitate PCa progression are unknown.We discovered that GCN2 is activated in a range of PCa cell lines, contributing to enhanced eIF2 phosphorylation and ATF4 expression. Genetic or pharmacological inhibition of GCN2 reduces growth in androgen‐sensitive and castration‐resistant PCa cell lines in culture and cell line‐derived and patient‐derived xenograft mouse models in vivo. Induction of GCN2 is accompanied by limitations of select amino acids and accumulation of cognate tRNAs that are reported to be activators of GCN2.A transcriptome analysis of PCa cells treated with a specific GCN2 small molecular inhibitor indicates that GCN2 is critical for expression of SLCgenes involved in metabolite transport. We found that GCN2 inhibition decreases intracellular amino acid levels accounting for reduced growth in PCa cells. Using CRISPR‐based phenotypic screens and genome‐wide gene expression analyses of wild‐type and GCN2‐depleted PCa cells, we confirmed the importance of the transporter genes in PCa fitness. One transporter, SLC3A2 (4F2), is induced by GCN2 and is essential for PCa proliferation. SLC3A2 engages with many nutrient transporters, allowing for their localization to the plasma membrane. Importantly, expression of SLC3A2 reduced GCN2 activation and rescued decreased amino acid levels and growth inhibition due to loss of GCN2.Our results indicate that select amino acid limitations activate GCN2 in PCa, resulting in the upregulation of key amino acid transporters, including 4F2 (SLC3A2), which provide for nutrient import to facilitate protein synthesis and metabolism required for PCa progression. We conclude that GCN2 and the ISR are promising therapeutic targets for both androgen‐sensitive and castration‐resistant prostate cancer.

The use of wet lab and computational docking experiments to elucidate idiosyncrasies associated with amino acid activation

Aminoacyl‐tRNA synthetases (aaRSs) are a well‐known class of enzymes that act as sentinels of genome. During protein translation, aaRSs ensure that L‐amino acids are charged or acylated onto their cognate tRNA isoacceptor(s). Hence, aaRSs help ensure that ribosomally translated proteins contain the proper sequence of amino acids. aaRS catalyze tRNA charging in a two‐step process. In step one, aaRSs selectively form a high energy amino acid adenylate. This step, refereed to amino acid activation, is the first level of selectivity in protein translation. Here, aaRSs effectively discriminate non‐cognate L‐amino acids. In step two, aaRSs transfer adenylated amino acids onto the cognate tRNA isoacceptor. This step is referred to as aminoacylation or tRNA charging. A great deal is known about aaRS reaction mechanisms but a number of confounding idiosyncrasies remain. The focus of this work aims to elucidate idiosyncrasies associated with amino acid activation. Here, a 32P‐based assay is used to measure the level to which E. coliaaRSs activate non‐cognate L‐amino acids. The purpose of these assays is to measure the plasticity of aaRS active sites to define additional underlying binding interactions that modulate amino acid recognition. This information could also be used to develop small molecule inhibitors of aaRS. Moreover, these data could reveal underlying clues to the potential origins of aaRSs. Here, three aaRSs are investigated: ArgRS, IleRS and LysRS. The activation data shows that each aaRS perhaps somewhat surprisingly activates more than five non‐cognate AAs to varying levels. Next, computer docking experiments are used to visualize the binding interaction of synthetase active site residues to each non‐cognate amino acid. These data are currently being used to construct protein‐ligand interaction maps of each aaRS active site. The resulting interactions maps can be used to more clearly elucidate idiosyncrasies associated with amino activation.

A polypeptide model for toxic aberrant proteins induced by aminoglycoside antibiotics.

Aminoglycoside antibiotics interfere with the selection of cognate tRNAs during translation, resulting in the synthesis of aberrant proteins that are the ultimate cause of cell death. However, the toxic potential of aberrant proteins and how they avoid degradation by the cell’s protein quality control (QC) machinery are not understood. Here we report that levels of the heat shock (HS) transcription factor σ32 increased sharply following exposure of Escherichia coli to the aminoglycoside kanamycin (Kan), suggesting that at least some of the aberrant proteins synthesized in these cells were recognized as substrates by DnaK, a molecular chaperone that regulates the HS response, the major protein QC pathway in bacteria. To further investigate aberrant protein toxic potential and interaction with cell QC factors, we studied an acutely toxic 48-residue polypeptide (ARF48) that is encoded by an alternate reading frame in a plant cDNA. As occurred in cells exposed to Kan, σ32 levels were strongly elevated following ARF48 expression, suggesting that ARF48 was recognized as a substrate by DnaK. Paradoxically, an internal 10-residue region that was tightly bound by DnaK in vitro also was required for the ARF48 toxic effect. Despite the increased levels of σ32, levels of several HS proteins were unchanged following ARF48 expression, suggesting that the HS response had been aborted. Nucleoids were condensed and cell permeability increased rapidly following ARF48 expression, together suggesting that ARF48 disrupts DNA-membrane interactions that could be required for efficient gene expression. Our results are consistent with earlier studies showing that aberrant proteins induced by aminoglycoside antibiotics disrupt cell membrane integrity. Insights into the mechanism for this effect could be gained by further study of the ARF48 model system.

Discovery of two distinct aminoacyl-tRNA synthetase complexes anchored to the Plasmodium surface tRNA import protein

Aminoacyl-tRNA synthetases (aaRSs) attach amino acids to their cognate transfer RNAs. In eukaryotes, a subset of cytosolic aaRSs is organized into a multisynthetase complex (MSC), along with specialized scaffolding proteins referred to as aaRS-interacting multifunctional proteins (AIMPs). In Plasmodium, the causative agent of malaria, the tRNA import protein (tRip), is a membrane protein that participates in tRNA trafficking; we show that tRip also functions as an AIMP. We identified three aaRSs, the glutamyl-tRNA synthetase (ERS), glutaminyl-tRNA synthetase (QRS), and methionyl-tRNA synthetase (MRS), which were specifically coimmunoprecipitated with tRip in Plasmodium berghei blood stage parasites. All four proteins contain an N-terminal glutathione-S-transferase (GST)–like domain that was demonstrated to be involved in MSC assembly. In contrast to previous studies, further dissection of GST-like interactions identified two exclusive heterotrimeric complexes: the Q-complex (tRip–ERS–QRS) and the M-complex (tRip–ERS–MRS). Gel filtration and light scattering suggest a 2:2:2 stoichiometry for both complexes but with distinct biophysical properties and mutational analysis further revealed that the GST-like domains of QRS and MRS use different strategies to bind ERS. Taken together, our results demonstrate that neither the singular homodimerization of tRip nor its localization in the parasite plasma membrane prevents the formation of MSCs in Plasmodium. Besides, the extracellular localization of the tRNA-binding module of tRip is compensated by the presence of additional tRNA-binding modules fused to MRS and QRS, providing each MSC with two spatially distinct functions: aminoacylation of intraparasitic tRNAs and binding of extracellular tRNAs. This unique host–pathogen interaction is discussed.

The Antiepileptic Valproic Acid Ameliorates Charcot-Marie-Tooth 2W (CMT2W) Disease-Associated HARS1 Mutation-Induced Inhibition of Neuronal Cell Morphological Differentiation Through c-Jun N-terminal Kinase.

Hereditary peripheral neuropathies called Charcot-Marie-Tooth (CMT) disease affect the sensory nerves as well as motor neurons. CMT diseases are composed of a heterogeneous group of diseases. They are characterized by symptoms such as muscle weakness and wasting. Type 2 CMT (CMT2) disease is a neuropathy with blunted or disrupted neuronal morphological differentiation phenotypes including process formation of peripheral neuronal axons. In the early stages of CMT2, demyelination that occurs in Schwann cells (glial cells) is rarely observed. CMT2W is an autosomal-dominant disease and is responsible for the gene encoding histidyl-tRNA synthetase 1 (HARS1), which is a family molecule of cytoplasmic aminoacyl-tRNA synthetases and functions by ligating histidine to its cognate tRNA. Despite increasing knowledge of the relationship of mutations on responsible genes with diseases, it still remains unclear how each mutation affects neuronal differentiation. Here we show that in neuronal N1E-115 cells, a severe Asp364-to-Tyr (D364Y) mutation of HARS1 leads to formation of small aggregates of HARS1 proteins; in contrast, wild type proteins are distributed throughout cell bodies. Expression of D364Y mutant proteins inhibited process formation whereas expression of wild type proteins possessed the normal differentiation ability to grow processes. Pretreatment with the antiepileptic valproic acid recovered inhibition of process formation by D364Y mutant proteins through the c-Jun N-terminal kinase signaling pathway. Taken together, these results indicate that the D364Y mutation of HARS1 causes HARS1 proteins to form small aggregates, inhibiting process growth, and that these effects are recovered by valproic acid. This could be a potential therapeutic drug for CMT2W at the cellular levels.

Eukaryotic tRNA sequences present conserved and amino acid-specific structural signatures.

Metazoan organisms have many tRNA genes responsible for decoding amino acids. The set of all tRNA genes can be grouped in sets of common amino acids and isoacceptor tRNAs that are aminoacylated by corresponding aminoacyl-tRNA synthetases. Analysis of tRNA alignments shows that, despite the high number of tRNA genes, specific tRNA sequence motifs are highly conserved across multicellular eukaryotes. The conservation often extends throughout the isoacceptors and isodecoders with, in some cases, two sets of conserved isodecoders. This study is focused on non-Watson–Crick base pairs in the helical stems, especially GoU pairs. Each of the four helical stems may contain one or more conserved GoU pairs. Some are amino acid specific and could represent identity elements for the cognate aminoacyl tRNA synthetases. Other GoU pairs are found in more than a single amino acid and could be critical for native folding of the tRNAs. Interestingly, some GoU pairs are anticodon-specific, and others are found in phylogenetically-specific clades. Although the distribution of conservation likely reflects a balance between accommodating isotype-specific functions as well as those shared by all tRNAs essential for ribosomal translation, such conservations may indicate the existence of specialized tRNAs for specific translation targets, cellular conditions, or alternative functions.

Evolutionary Adjustment of tRNA Identity Rules in Bacillariophyta for Recognition by an Aminoacyl-tRNA Synthetase Adds a Facet to the Origin of Diatoms

Error-free protein synthesis relies on the precise recognition by the aminoacyl-tRNA synthetases of their cognate tRNAs in order to attach the corresponding amino acid. A concept of universal tRNA identity elements requires the aminoacyl-tRNA synthetases provided by the genome of an organism to match the identity elements found in the cognate tRNAs in an evolution-independent manner. Identity elements tend to cluster in the tRNA anticodon and acceptor stem regions. However, in the arginine system, in addition to the anticodon, the importance of nucleotide A20 in the tRNA D-loop for cognate enzyme recognition has been a sustained feature for arginyl-tRNA synthetase in archaea, bacteria and in the nuclear-encoded cytosolic form in mammals and plants. However, nuclear-encoded mitochondrial arginyl-tRNA synthetase, which can be distinguished from its cytosolic form by the presence or absence of signature motifs, dispenses with the A20 requirement. An examination of several hundred non-metazoan organisms and their corresponding tRNAArg substrates has confirmed this general concept to a large extent and over numerous phyla. However, some Stramenopiles, and in particular, Diatoms (Bacillariophyta) present a notable exception. Unusually for non-fungal organisms, the nuclear genome encodes tRNAArg isoacceptors with C or U at position 20. In this case one of two nuclear-encoded cytosolic arginyl-tRNA synthetases has evolved to become insensitive to the nature of the D-loop identity element. The other, with a binding pocket that is compatible with tRNAArg-A20 recognition, is targeted to organelles that encode solely such tRNAs.

Leucyl-tRNA synthetase is a tumour suppressor in breast cancer and regulates codon-dependent translation dynamics.

Tumourigenesis and cancer progression require enhanced global protein translation1–3. Such enhanced translation is caused by oncogenic and tumour suppressive events that drive the synthesis and activity of translational machinery4,5. Here we report the surprising observation that leucyl-tRNA synthetase (LARS) becomes repressed during mammary cell transformation and in human breast cancer. Monoallelic genetic deletion of LARS in mouse mammary glands enhanced breast cancer tumour formation and proliferation. LARS repression reduced the abundance of select leucine tRNA isoacceptors, leading to impaired leucine codon-dependent translation of growth suppressive genes including epithelial membrane protein 3 (EMP3) and gamma-glutamyltransferase 5 (GGT5). Our findings uncover a tumour suppressive tRNA synthetase and reveal that dynamic repression of a specific tRNA synthetase—along with its downstream cognate tRNAs—elicits a downstream codon-biased translational gene network response that enhances breast tumour formation and growth.

Directed Evolution Pipeline for the Improvement of Orthogonal Translation Machinery for Genetic Code Expansion at Sense Codons.

The expansion of the genetic code beyond a single type of noncanonical amino acid (ncAA) is hindered by inefficient machinery for reassigning the meaning of sense codons. A major obstacle to using directed evolution to improve the efficiency of sense codon reassignment is that fractional sense codon reassignments lead to heterogeneous mixtures of full-length proteins with either a ncAA or a natural amino acid incorporated in response to the targeted codon. In stop codon suppression systems, missed incorporations lead to truncated proteins; improvements in activity may be inferred from increased protein yields or the production of downstream reporters. In sense codon reassignment, the heterogeneous proteins produced greatly complicate the development of screens for variants of the orthogonal machinery with improved activity. We describe the use of a previously-reported fluorescence-based screen for sense codon reassignment as the first step in a directed evolution workflow to improve the incorporation of a ncAA in response to the Arg AGG sense codon. We first screened a library with diversity introduced into both the orthogonal Methanocaldococcus jannaschii tyrosyl tRNA anticodon loop and the cognate aminoacyl tRNA synthetase (aaRS) anticodon binding domain for variants that improved incorporation of tyrosine in response to the AGG codon. The most efficient variants produced fluorescent proteins at levels indistinguishable from the E. coli translation machinery decoding tyrosine codons. Mutations to the M. jannaschii aaRS that were found to improve tyrosine incorporation were transplanted onto a M. jannaschii aaRS evolved for the incorporation of para-azidophenylalanine. Improved ncAA incorporation was evident using fluorescence- and mass-based reporters. The described workflow is generalizable and should enable the rapid tailoring of orthogonal machinery capable of activating diverse ncAAs to any sense codon target. We evaluated the selection based improvements of the orthogonal pair in a host genomically engineered for reduced target codon competition. Using this particular system for evaluation of arginine AGG codon reassignment, however, E. coli strains with genomes engineered to remove competing tRNAs did not outperform a standard laboratory E. coli strain in sense codon reassignment.

Gain of C-Ala enables AlaRS to target the L-shaped tRNAAla.

Unlike many other aminoacyl-tRNA synthetases, alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout biology. While Caenorhabditis elegans cytoplasmic AlaRS (CeAlaRSc) retains the prototype structure, its mitochondrial counterpart (CeAlaRSm) contains only a residual C-terminal domain (C-Ala). We demonstrated herein that the C-Ala domain from CeAlaRSc robustly binds both tRNA and DNA. It bound different tRNAs but preferred tRNAAla. Deletion of this domain from CeAlaRSc sharply reduced its aminoacylation activity, while fusion of this domain to CeAlaRSm selectively and distinctly enhanced its aminoacylation activity toward the elbow-containing (or L-shaped) tRNAAla. Phylogenetic analysis showed that CeAlaRSm once possessed the C-Ala domain but later lost most of it during evolution, perhaps in response to the deletion of the T-arm (part of the elbow) from its cognate tRNA. This study underscores the evolutionary gain of C-Ala for docking AlaRS to the L-shaped tRNAAla.

Amino Acid Specificity of Ancestral Aminoacyl-tRNA Synthetase Prior to the Last Universal Common Ancestor Commonote commonote

Extant organisms commonly use 20 amino acids in protein synthesis. In the translation system, aminoacyl-tRNA synthetase (ARS) selectively binds an amino acid and transfers it to the cognate tRNA. It is postulated that the amino acid repertoire of ARS expanded during the development of the translation system. In this study we generated composite phylogenetic trees for seven ARSs (SerRS, ProRS, ThrRS, GlyRS-1, HisRS, AspRS, and LysRS) which are thought to have diverged by gene duplication followed by mutation, before the evolution of the last universal common ancestor. The composite phylogenetic tree shows that the AspRS/LysRS branch diverged from the other five ARSs at the deepest node, with the GlyRS/HisRS branch and the other three ARSs (ThrRS, ProRS and SerRS) diverging at the second deepest node. ThrRS diverged next, and finally ProRS and SerRS diverged from each other. Based on the phylogenetic tree, sequences of the ancestral ARSs prior to the evolution of the last universal common ancestor were predicted. The amino acid specificity of each ancestral ARS was then postulated by comparison with amino acid recognition sites of ARSs of extant organisms. Our predictions demonstrate that ancestral ARSs had substantial specificity and that the number of amino acid types amino-acylated by proteinaceous ARSs was limited before the appearance of a fuller range of proteinaceous ARS species. From an assumption that 10 amino acid species are required for folding and function, proteinaceous ARS possibly evolved in a translation system composed of preexisting ribozyme ARSs, before the evolution of the last universal common ancestor.

The Landscape of Aminoacyl-tRNA Synthetases Involved in Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in translation by linking amino acids onto their cognate tRNAs during protein synthesis. During evolution, aaRSs develop numerous non-canonical functions that expand the roles of aaRSs in eukaryotic organisms. Although aaRSs have been implicated in viral infection, the function of aaRSs during infections with coronaviruses (CoVs) remains unclear. Here, we analyzed the data from transcriptomic and proteomic database on human cytoplasmic (cyto) and mitochondrial (mt) aaRSs across infections with three highly pathogenic human CoVs, with a particular focus on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We found an overall downregulation of aaRSs at mRNA levels, while the protein levels of some mt-aaRSs and the phosphorylation of certain aaRSs were increased in response to SARS-CoV-2 infection. Strikingly, interaction network between SARS-CoV-2 and human aaRSs displayed a strong involvement of mt-aaRSs. Further co-immunoprecipitation (co-IP) experiments confirmed the physical interaction between SARS-CoV-2 M protein and TARS2. In addition, we identified the intermediate nodes and potential pathways involved in SARS-CoV-2 infection. This study provides an unbiased, overarching perspective on the correlation between aaRSs and SARS-CoV-2. More importantly, this work identifies TARS2, HARS2, and EARS2 as potential key factors involved in COVID-19.

Dynamics of the Catalytic Active Site of Isoleucyl tRNA Synthetase from Staphylococcus aureus bound with Adenylate and Mupirocin.

The development of new antimicrobial drugs is critically needed due to the alarming increase in antibiotic resistance in bacterial pathogens. The active sites of different bacterial aminoacyl tRNA synthetases (aaRS) are validated targets of antibiotics. At present, the only aaRS inhibitor approved is mupirocin (MRC) which targets bacterial isoleucyl tRNA synthetase (IleRS). The present work is aimed at understanding the lacunae of knowledge concerning the active site conformational dynamics in IleRS in the presence of inhibitor mupirocin. With this end in view, we have carried out classical molecular dynamics simulation and metadynamics simulations of the open state of IleRS from Staphylococcus aureus (SaIleRS), the closed state tripartite complex bound with cognate adenylate (Ile-AMP) and tRNA, the closed state tripartite complex bound with noncognate MRC, and the closed state tripartite complex bound with tRNA and MRC with mutated SaIleRS (V588F). The present simulation established a dynamic picture of SaIleRS complexed with cognate and the noncognate substrates which are completely consistent with crystallographic and biochemical studies and explain the existing lacunae of knowledge. The active site is significantly more compact in the Ile-AMP bound complex compared to the open state due to the closure of the KMSKS and HMGH loops and clamping down of the tRNA acceptor end near the active site gate. The present result shows that the unusual open conformational state of the KMSKS loop observed in the cognate substrate-bound complex in the crystal is due to crystallographic constraints. Although the mupirocin tightly fits the catalytic active site in the MRC-bound complex, the nonanoic acid moiety is partly exposed to the water. The KMSKS loop is pushed open in the MRC-bound complex to accommodate the noncognate MRC. New tunnels open up, extending to the editing site in the complex. Out of its three broad segments, the C12 to C17 segment, the conjugated segment, and the nonanoic moiety, the conjugated part of MRC binds most effectively with the active site of the MRC-bound complex. The aromatic residues packing around the C12 to C17 segment of MRC stabilize the tRNA hairpin conformation in a similar way as observed in the TrpRS. The V588F mutation is weakening the interaction between this region of the active site and weakens the binding of MRC in the active site. This result explains why the V588F mutation is responsible for low-level mupirocin resistance. The free energy of unbinding the conjugated segment (and C12 to C17 segment, as well) largely contributes to the total free energy of unbinding the MRC. The active site organization of IleRS from eukaryotic Candida albicans is compared with the bacterial IleRS active site to understand the low binding affinity in eukaryotic IleRS. The present study could be a starting point of future studies related to the development of effective drug binding in the SaIleRS.

Indirect Routes to Aminoacyl-tRNA: The Diversity of Prokaryotic Cysteine Encoding Systems.

Universally present aminoacyl-tRNA synthetases (aaRSs) stringently recognize their cognate tRNAs and acylate them with one of the proteinogenic amino acids. However, some organisms possess aaRSs that deviate from the accurate translation of the genetic code and exhibit relaxed specificity toward their tRNA and/or amino acid substrates. Typically, these aaRSs are part of an indirect pathway in which multiple enzymes participate in the formation of the correct aminoacyl-tRNA product. The indirect cysteine (Cys)-tRNA pathway, originally thought to be restricted to methanogenic archaea, uses the unique O-phosphoseryl-tRNA synthetase (SepRS), which acylates the non-proteinogenic amino acid O-phosphoserine (Sep) onto tRNACys. Together with Sep-tRNA:Cys-tRNA synthase (SepCysS) and the adapter protein SepCysE, SepRS forms a transsulfursome complex responsible for shuttling Sep-tRNACys to SepCysS for conversion of the tRNA-bound Sep to Cys. Here, we report a comprehensive bioinformatic analysis of the diversity of indirect Cys encoding systems. These systems are present in more diverse groups of bacteria and archaea than previously known. Given the occurrence and distribution of some genes consistently flanking SepRS, it is likely that this gene was part of an ancient operon that suffered a gradual loss of its original components. Newly identified bacterial SepRS sequences strengthen the suggestion that this lineage of enzymes may not rely on the m1G37 identity determinant in tRNA. Some bacterial SepRSs possess an N-terminal fusion resembling a threonyl-tRNA synthetase editing domain, which interestingly is frequently observed in the vicinity of archaeal SepCysS genes. We also found several highly degenerate SepRS genes that likely have altered amino acid specificity. Cross-analysis of selenocysteine (Sec)-utilizing traits confirmed the co-occurrence of SepCysE and the Sec-utilizing machinery in archaea, but also identified an unusual O-phosphoseryl-tRNASec kinase fusion with an archaeal Sec elongation factor in some lineages, where it may serve in place of SepCysE to prevent crosstalk between the two minor aminoacylation systems. These results shed new light on the variations in SepRS and SepCysS enzymes that may reflect adaptation to lifestyle and habitat, and provide new information on the evolution of the genetic code.

Conformational sampling of CMT-2D associated GlyRS mutations

During protein synthesis, aminoacyl-tRNA synthetases covalently link amino acids with their cognate tRNAs. Amino acid mutations in glycyl-tRNA synthetase can disrupt protein synthesis and lead to a neurological disorder known as Charcot-Marie-Tooth disease type 2D (CMT-2D). Several studies employing diverse techniques have identified potential disease mechanisms at the molecular level. The majority of CMT-2D mutations in glycyl-tRNA are found within its dimer interface. However, no atomic structures bearing these mutations have been solved. Consequently, the specific disease-causing structural changes that occur in glycyl-tRNA synthetase have not been definitively established. Here we use molecular dynamics simulations to probe conformational changes in glycyl-tRNA synthetase caused by one mutation within the dimer interface: G240R. Our results show that the mutation alters the number of native interactions at the dimer interface and also leads to altered dynamics of two regions of glycyl-tRNA synthetase associated with tRNA binding. Additionally, we use our simulations to make predictions about the effects of other clinically reported CMT-2D mutations. Our results identify a region of the glycyl-tRNA synthetase structure that may be disrupted in a large number of CMT-2D mutations. Structural changes in this region may be a common molecular mechanism in glycyl-tRNA synthetase CMT-2D pathologies. Statement of significanceIn this study, we use molecular dynamics simulations to elucidate structural conformations accessible to glycyl-tRNA synthetase (GlyRS), an enzyme that ligates cytosolic glycine with tRNA-Gly. This protein contains multiple flexible regions with dynamics that elude in vitro structural characterization. Our computational approach provides unparalleled atomistic details of structural changes in GlyRS that contribute to its role in protein synthesis. A number of mutations in GlyRS are associated with a peripheral nerve disorder, Charcot-Marie-Tooth disease type 2D (CMT-2D). Mutation-induced structural and dynamic changes in GlyRS have similarity that elude in vitro structural characterization. Our simulations provide insights into disease mechanisms for one such mutation: G240R. Additionally, we leverage our computational data to identify regions of GlyRS critical to its function and to predict the effects of other disease-associated mutations. These results open up new directions for research into the molecular characterization of GlyRS and into hypothesis-driven studies of CMT-2D disease mechanisms.

Predicted Functional Shifts Due to Type of Soil Microbiome and Watering of Two Wild Plants in Western Region of Saudi Arabia

The present study aimed to predict differential enrichment of pathways and compounds in the rhizosphere microbiomes of the two wild plants (Abutilon fruticosum and Nitrosalsola vermiculata) and to predict functional shifts in microbiomes due to water. Amplicon sequencing of 16S rRNA region V3–V4 was done and gene-based microbial compositions were enrolled in PICRUSt to predict enriched pathways and compounds. The results indicated that “ABC transporters” and “Quorum sensing” pathways are among the highest enriched pathways in rhizosphere microbiomes of the two wild plants compared with those of the bulk soil microbiomes. The highest enriched compounds in soil microbiomes of the two wild plants included five proteins and three enzymes participating in one or more KEGG pathways. Six of these eight compounds showed higher predicted enrichment in rhizosphere soil microbiomes, while only one, namely phosphate transport system substrate-binding protein, showed higher enrichment in the surrounding bulk soil microbiomes. In terms of differentially enriched compounds due to watering, only the dual-specific aspartyl-tRNA (Asn)/glutamyl-tRNA (Gln) amidotransferase subunit A showed higher enrichment in rhizosphere soil of the two wild plants after 24 h of watering. Two of the highly enriched compounds namely branched-chain amino acid transport system ATP-binding protein and branched-chain amino acid transport system substrate-binding protein, are encoded by genes stimulated by the plant’s GABA that participates in conferring biotic and abiotic stresses in plants and improves the plant’s growth performance. The 3-Oxoacyl-[ACP] reductase, a member of the short-chain alcohol dehydrogenase/ reductase (SDR) superfamily, participates in fatty acids elongation cycles and contributes to plant-microbe symbiotic relationships, while enoyl-CoA hydratase has a reverse action as it participates in “Fatty acid degradation” pathway. The methyl-accepting chemotaxis protein is an environmental signal that sense “Bacterial chemotaxis” pathway to help establishing symbiosis with plant roots by recruiting/colonizing of microbial partners (symbionts) to plant rhizosphere. This information justifies the high enrichment of compounds in plant rhizosphere. The dual-specific aspartyl-tRNA (Asn)/glutamyl-tRNA (Gln) amidotransferase subunit A contributes to the plant ability to respond to watering as it participates in attaching the correct amino acid during translation to its cognate tRNA species, while hydrolyzing incorrectly attached amino acid. These two actions reduce the influence of oxidative stress in generating misfolded proteins and in reducing fidelity of translation.

Directed-evolution of translation system for efficient unnatural amino acids incorporation and generalizable synthetic auxotroph construction

Site-specific incorporation of unnatural amino acids (UAAs) with similar incorporation efficiency to that of natural amino acids (NAAs) and low background activity is extremely valuable for efficient synthesis of proteins with diverse new chemical functions and design of various synthetic auxotrophs. However, such efficient translation systems remain largely unknown in the literature. Here, we describe engineered chimeric phenylalanine systems that dramatically increase the yield of proteins bearing UAAs, through systematic engineering of the aminoacyl-tRNA synthetase and its respective cognate tRNA. These engineered synthetase/tRNA pairs allow single-site and multi-site incorporation of UAAs with efficiencies similar to those of NAAs and high fidelity. In addition, using the evolved chimeric phenylalanine system, we construct a series of E. coli strains whose growth is strictly dependent on exogenously supplied of UAAs. We further show that synthetic auxotrophic cells can grow robustly in living mice when UAAs are supplemented.

Systematic expression profiling of neuropathy-related aminoacyl-tRNA synthetases in zebrafish during development

Aminoacyl tRNA synthetases (ARSs) are a group of proteins, acting as transporters to transfer and attach the appropriate amino acids onto their cognate tRNAs for translation. So far, 18 out of 20 cytoplasmic ARSs are reported to be connected to different neuropathy disorders with multi-organ defects that are often accompanied with developmental delays. Thus, it is important to understand functions and impacts of ARSs at the whole organism level. Here, we systematically analyzed the spatiotemporal expression of 14 ars and 2 aimp genes during development in zebrafish that have not be previously reported. Not only in the brain, their dynamic expression patterns in several tissues such as in the muscles, liver and intestine suggest diverse roles in a wide range of development processes in addition to neuronal function, which is consistent with potential involvement in multiple syndrome diseases associated with ARS mutations. In particular, hinted by its robust expression pattern in the brain, we confirmed that aimp1 is required for the formation of cerebrovasculature by a loss-of-function approach. Overall, our systematic profiling data provides a useful basis for studying roles of ARSs during development and understanding their potential functions in the etiology of related diseases.

Decoding the impact of disease-causing mutations in an essential aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases are housekeeping enzymes that catalyze the specific attachment of amino acids onto cognate tRNAs, providing building blocks for ribosomal protein synthesis. Owing to the absolutely essential nature of these enzymes, the possibility that mutations in their sequence could be the underlying cause of diseases had not been foreseen. However, we are learning of patients bearing familial mutations in aminoacyl-tRNA synthetases at an exponential rate. In a recent issue of JBC, Jin et al. analyzed the impact of two such mutations in the very special bifunctional human glutamyl-prolyl-tRNA synthetase and convincingly decode how these mutations elicit the integrated stress response.