threonyl-tRNA Synthetase Research Articles

Identifying the targets of aminoacyl-tRNA synthetase inhibitors by primer extension inhibition

Aminoacyl-transfer RNA (tRNA) synthetases (RS) are essential components of the cellular translation machinery and can be exploited for antibiotic discovery. Because cells have many different RS, usually one for each amino acid, identification of the specific enzyme targeted by a new natural or synthetic inhibitor can be cumbersome. We describe the use of the primer extension technique in conjunction with specifically designed synthetic genes to identify the RS targeted by an inhibitor. Suppression of a synthetase activity reduces the amount of the cognate aminoacyl-tRNA in a cell-free translation system resulting in arrest of translation when the corresponding codon enters the decoding center of the ribosome. The utility of the technique is demonstrated by identifying a switch in target specificity of some synthetic inhibitors of threonyl-tRNA synthetase.

Abstract 3897: A novel function for threonyl-tRNA synthetase as a stimulator of angiogenesis and endothelial cell migration.

Abstract In mammalian systems, several aminoacyl-tRNA synthetases are known to have distinct non-canonical functionality. For the first time, we demonstrate that human threonyl-tRNA synthetase (TARS) induces angiogenesis through endothelial cell signaling, and this induction can be blocked by the borrelidin derivative compound BC194. Upon stimulation with vascular endothelial growth factor (VEGF) or tumor necrosis factor-alpha (TNF-α), human endothelial cells secrete TARS into the extracellular space. Extracellular TARS induces and BC194 inhibits angiogenesis in both in vitro tube formation assays and in vivo chick chorioallantoic membrane assays. Furthermore, the anti-angiogenic effects of BC194 are specific to its inhibition of TARS angiogenic activity rather than an indirect stress response. Additional evidence supports a stimulatory role for TARS in endothelial cell migration as a mechanism for its angiogenic activity. Taken together, these findings reveal a previously undefined function for TARS as an angiogenesis inducer stimulated by hypoxic and inflammatory signals. Detecting or intervening in the non-canonical extracellular TARS signaling pathway may thus provide a novel means of diagnosing or treating angiogenesis and inflammatory responses involved in cancer. Citation Format: Tamara Williams, Adam Mirando, Christopher Francklyn, Karen M. Lounsbury. A novel function for threonyl-tRNA synthetase as a stimulator of angiogenesis and endothelial cell migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3897. doi:10.1158/1538-7445.AM2013-3897

Secreted Threonyl-tRNA synthetase stimulates endothelial cell migration and angiogenesis

Aminoacyl-tRNA synthetases classically regulate protein synthesis but some also engage in alternative signaling functions related to immune responses and angiogenesis. Threonyl-tRNA synthetase (TARS) is an autoantigen in the autoimmune disorder myositis, and borrelidin, a potent inhibitor of TARS, inhibits angiogenesis. We explored a mechanistic link between these findings by testing whether TARS directly affects angiogenesis through inflammatory mediators. When human vascular endothelial cells were exposed to tumor necrosis factor-α (TNF-α) or vascular endothelial growth factor (VEGF), TARS was secreted into the cell media. Furthermore, exogenous TARS stimulated endothelial cell migration and angiogenesis in both in vitro and in vivo assays. The borrelidin derivative BC194 reduced the angiogenic effect of both VEGF and TARS, but not a borrelidin-resistant TARS mutant. Our findings reveal a previously undiscovered function for TARS as an angiogenic, pro-migratory extracellular signaling molecule. TARS thus provides a potential target for detecting or interdicting disease-related inflammatory or angiogenic responses.

Identification of Bacteria-Selective Threonyl-tRNA Synthetase Substrate Inhibitors by Structure-Based Design

A series of potent and bacteria-selective threonyl-tRNA synthetase (ThrRS) inhibitors have been identified using structure-based drug design. These compounds occupied the substrate binding site of ThrRS and showed excellent binding affinities for all of the bacterial orthologues tested. Some of the compounds displayed greatly improved bacterial selectivity. Key residues responsible for potency and bacteria/human ThrRS selectivity have been identified. Antimicrobial activity has been achieved against wild-type Haemophilus influenzae and efflux-deficient mutants of Escherichia coli and Burkholderia thailandensis.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analyses of threonyl-tRNA synthetase editing domain fromAeropyrum pernix

The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeon Pyrococcus abysii in complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms. To probe further into the mechanistic and evolutionary aspects of this domain, work has been extended to another archaeon Aeropyrum pernix. The organism possesses two proteins corresponding to threonyl-tRNA synthetase, i.e. ThrRS1 and ThrRS2, encoded by two different genes, thrS1 and thrS2, respectively. ThrRS1 is responsible for aminoacylation and ThrRS2 for proofreading activity. Here the purification, crystallization and preliminary X-ray crystallographic investigation of the N-terminal proofreading domain of ThrRS2 from A. pernix is reported. The crystals belong to either the P4(1)2(1)2 or P4(3)2(1)2 space group and consist of one monomer per asymmetric unit.

Translational fidelity maintenance preventing Ser mis-incorporation at Thr codon in protein from eukaryote

Aminoacyl-tRNA synthetase (aaRS) catalyzes the first step of protein synthesis, producing aminoacyl-tRNAs as building blocks. Eukaryotic aaRS differs from its prokaryotic counterpart in terminal extension or insertion. Moreover, the editing function of aaRSs is an indispensable checkpoint excluding non-cognate amino acids at a given codon and ensuring overall translational fidelity. We found higher eukaryotes encode two cytoplasmic threonyl-tRNA synthetases (ThrRSs) with difference in N-terminus. The longer isoform is more closely related to the ThrRSs of higher eukaryotes than to those of lower eukaryotes. A yeast strain was generated to include deletion of the thrS gene encoding ThrRS. Combining in vitro biochemical and in vivo genetic data, ThrRSs from eukaryotic cytoplasm were systematically analyzed, and role of the eukaryotic cytoplasmic ThrRS-specific N-terminal extension was elucidated. Furthermore, the mechanisms of aminoacylation and editing activity mediated by Saccharomyces cerevisiae ThrRS (ScThrRS) were clarified. Interestingly, yeast cells were tolerant of variation at the editing active sites of ScThrRS without significant Thr-to-Ser conversion in the proteome even under significant environmental stress, implying checkpoints downstream of aminoacylation to provide a further quality control mechanism for the yeast translation system. This study has provided the first comprehensive elucidation of the translational fidelity control mechanism of eukaryotic ThrRS.

Borrelidin, a Potent Antifungal Agent: Insight into the Antifungal Mechanism against Phytophthora sojae

Borrelidin has high and specific antifungal activity against Phytophthora sojae . To explore the antifungal mechanism of borrelidin against P. sojae , the relationship between the antifungal activity of borrelidin and the concentration of threonine was evaluated. The results demonstrated that the growth-inhibitory effect of borrelidin on the growth of P. sojae was antagonized by threonine in a dose-dependent manner, suggesting that threonyl-tRNA synthetase (ThrRS) may be the potential target of borrelidin. Subsequently, the inhibition of the enzymatic activity of ThrRS by borrelidin in vitro was confirmed. Furthermore, the detailed interaction between ThrRS and borrelidin was investigated using fluorescence spectroscopy and circular dichroism (CD), implying a tight binding of borrelidin to ThrRS. Taken together, these results suggest that the antifungal activity of borrelidin against P. sojae was mediated by inhibition of ThrRS via the formation of the ThrRS-borrelidin complex.

The Mechanism of Pre-transfer Editing in Yeast Mitochondrial Threonyl-tRNA Synthetase

Accurate translation of mRNA into protein is a fundamental biological process critical for maintaining normal cellular functions. To ensure translational fidelity, aminoacyl-tRNA synthetases (aaRSs) employ pre-transfer and post-transfer editing activities to hydrolyze misactivated and mischarged amino acids, respectively. Whereas post-transfer editing, which requires either a specialized domain in aaRS or a trans-protein factor, is well described, the mechanism of pre-transfer editing is less understood. Here, we show that yeast mitochondrial threonyl-tRNA synthetase (MST1), which lacks an editing domain, utilizes pre-transfer editing to discriminate against serine. MST1 misactivates serine and edits seryl adenylate (Ser-AMP) in a tRNA-independent manner. MST1 hydrolyzes 80% of misactivated Ser-AMP at a rate 4-fold higher than that for the cognate threonyl adenylate (Thr-AMP) while releasing 20% of Ser-AMP into the solution. To understand the mechanism of pre-transfer editing, we solved the crystal structure of MST1 complexed with an analog of Ser-AMP. The binding of the Ser-AMP analog to MST1 induces conformational changes in the aminoacylation active site, and it positions a potential hydrolytic water molecule more favorably for nucleophilic attack. In addition, inhibition results reveal that the Ser-AMP analog binds the active site 100-fold less tightly than the Thr-AMP analog. In conclusion, we propose that the plasticity of the aminoacylation site in MST1 allows binding of Ser-AMP and the appropriate positioning of the hydrolytic water molecule.

Molecular Dynamics Investigation into Substrate Binding and Identity of the Catalytic Base in the Mechanism of Threonyl-tRNA Synthetase

The structure and nature of the fully bound active site of Threonyl-tRNA Synthetase (ThrRS) for the second half-reaction has been investigated using molecular dynamics simulations. More specifically, we examined the ThrRS active site with both the substrate Threonyl-AMP and the cosubstrate cognate Threonyl-tRNA bound. Furthermore, we also considered the cases in which an active-site histidyl residue (His309) is either neutral or protonated. Moreover, we considered the role a water molecule may play in formation of a viable Michaelis complex. From the results it is found that the most likely role of His309 is in binding and properly orientating the ribose of the Ado76 nucleotidyl residue of the threonyl-tRNA via formation of a direct His309···Ado76 hydrogen bond, i.e., without involvement of a water. In addition, the imidazole of the His309 residue is likely neutral. It was found that upon protonation the positioning of the Ado76-3'-OH was perturbed, leading to a reduced chance for nucleophilic attack of the threonyl's C1 center.

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

Aminoacyl-tRNA synthetases (aaRSs) ensure faithful translation of mRNA into protein by coupling an amino acid to a set of tRNAs with conserved anticodon sequences. Here, we show that in mitochondria of Saccharomyces cerevisiae, a single aaRS (MST1) recognizes and aminoacylates two natural tRNAs that contain anticodon loops of different size and sequence. Besides a regular tRNA(2Thr) with a threonine (Thr) anticodon, MST1 also recognizes an unusual tRNA(1Thr), which contains an enlarged anticodon loop and an anticodon triplet that reassigns the CUN codons from leucine to threonine. Our data show that MST1 recognizes the anticodon loop in both tRNAs, but employs distinct recognition mechanisms. The size but not the sequence of the anticodon loop is critical for tRNA(1Thr) recognition, whereas the anticodon sequence is essential for aminoacylation of tRNA(2Thr). The crystal structure of MST1 reveals that, while lacking the N-terminal editing domain, the enzyme closely resembles the bacterial threonyl-tRNA synthetase (ThrRS). A detailed structural comparison with Escherichia coli ThrRS, which is unable to aminoacylate tRNA(1Thr), reveals differences in the anticodon-binding domain that probably allow recognition of the distinct anticodon loops. Finally, our mutational and modeling analyses identify the structural elements in MST1 (e.g., helix α11) that define tRNA selectivity. Thus, MTS1 exemplifies that a single aaRS can recognize completely divergent anticodon loops of natural isoacceptor tRNAs and that in doing so it facilitates the reassignment of the genetic code in yeast mitochondria.

Uneven spread of cis- and trans-editing aminoacyl-tRNA synthetase domains within translational compartments of P. falciparum.

Accuracy of aminoacylation is dependent on maintaining fidelity during attachment of amino acids to cognate tRNAs. Cis- and trans-editing protein factors impose quality control during protein translation, and 8 of 36 Plasmodium falciparum aminoacyl-tRNA synthetase (aaRS) assemblies contain canonical putative editing modules. Based on expression and localization profiles of these 8 aaRSs, we propose an asymmetric distribution between the parasite cytoplasm and its apicoplast of putative editing-domain containing aaRSs. We also show that the single copy alanyl- and threonyl-tRNA synthetases are dually targeted to parasite cytoplasm and apicoplast. This bipolar presence of two unique synthetases presents opportunity for inhibitor targeting their aminoacylation and editing activities in twin parasite compartments. We used this approach to identify specific inhibitors against the alanyl- and threonyl-tRNA synthetases. Further development of such inhibitors may lead to anti-parasitics which simultaneously block protein translation in two key parasite organelles, a strategy of wider applicability for pathogen control.

Occurrence, isolation and DNA identification of Streptococcus thermophilus involved in Algerian traditional butter ‘Smen’

Streptococcus thermophilus isolates from traditional butter 'Smen', a fermented product from cow's and ewe's milk in arid area was subjected to taxonomical investigations. The identification procedure included phenotypic approaches, molecular characterization by using genus polymerase chain reaction (PCR) amplifications for sodA gene encoding the manganese-dependant superoxide dismutase A, and species-specific primers from gene encoding glucose kinase (glcK), gene encoding DNA polymerase III (dnaE) and gene encoding threonyl-tRNA synthetase (thrS) housekeeping genes in order to distinguish among reference, and wild strains of S. thermophilus and for their differentiation from Enterococcus spp. A total of 12 strains were tested by DNA identification analysis and these indigenous isolates were unambiguously characterised by their housekeeping gene profiles. Finally, four genotypes were recognised. To our knowledge, this is the first report on the isolation and molecular characterization of S. thermophilus strains from fermented milk transformed into home-production butter provided from Southern West Algeria. These results are of particular interest as they favour the selection of future cultures starters as S. thermophilus from traditional dairy products. It is, therefore, of great importance to preserve the genetic pool of the wild strains.

Clinical heterogeneity and prognostic features of South Australian patients with anti‐synthetase autoantibodies

To determine the clinical, serological and prognostic features of patients with autoantibodies against three aminoacyl-transfer RNA synthetases (ARS), namely Jo-1 (histidyl-tRNA synthetase), PL-7 (threonyl-tRNA synthetase) and PL-12 (alanyl-tRNA synthetase) in South Australia. Patients with autoantibodies against ARS detected by line immunoassay (anti-Jo1, anti-PL7, anti-PL12) or enzyme-linked immunosorbent assay (anti-Jo1) were identified from existing laboratory databases for the period 1994-2009. Demographic, clinical and serological data were obtained by retrospective review of patients' medical records and laboratory databases. Forty-two patients with autoantibodies were identified (anti-Jo1 = 37, anti-PL7 = 4, anti-PL12 = 1). Females were more commonly affected than males (M : F = 12:30). Twenty-one patients had polymyositis (anti-Jo1 = 17, anti-PL7 = 4), seven dermatomyositis (anti-Jo1 = 6, anti-PL12 = 1), 10 overlap syndrome (anti-Jo1 = 10; lupus = 4, scleroderma = 3, Sjögren's syndrome = 2 and rheumatoid arthritis = 2) and four had interstitial lung disease (ILD) only (anti-Jo1 = 4). ILD was present in 69%, polyarthritis in 59% and positive anti-nuclear antibody (ANA) in 43% of patients. Concurrence of autoantibodies against Ro-52 with Jo-1 was seen in 12 patients. The mean follow-up period was 8.3 years (95% CI 5.8-10.8) with 12 deaths. Poor prognostic indicators were age of onset >60 years (P= 0.001), cancer (P= 0.002), negative ANA (P= 0.006) and negative autoantibodies to extractable nuclear antigens (P= 0.02). Patients with autoantibodies against ARS present with varied clinical features and occasionally with isolated lung involvement (amyopathic ILD). Older age of onset, malignancy and negative immunologic tests are predictors of poor prognosis. Concurrence of autoantibodies against Jo-1 and Ro-52 may reflect a coupling effect during generation of autoimmunity.

Borrelidin, a small molecule nitrile-containing macrolide inhibitor of threonyl-tRNA synthetase, is a potent inducer of apoptosis in acute lymphoblastic leukemia

Due to the poor prognosis and limited therapeutic options for adult patients with acute lymphoblastic leukemia (ALL), development of novel therapies is much needed to prolong patient survival and increase the efficacy of their treatment. Malignant T cells need high levels of nutrients to maintain their proliferation rate. Borrelidin, a small molecule nitrile-containing macrolide, is an inhibitor of bacterial and eukaryal threonyl-tRNA synthetase. Borrelidin-mediated inhibition of aminoacyl-tRNA synthesis, leads to an induction in the levels of uncharged tRNA, nutritional stress and ultimately inhibition of protein synthesis. The aim of the present study was to investigate whether borrelidin treatment inhibits the proliferation of malignant ALL cell lines, Jurkat and CEM cells, and study the mechanism by which this drug acts. Our results show that borrelidin was able to potently inhibit the proliferation of ALL cell lines with a half maximal inhibitory concentration of 50ng/ml. Borrelidin showed a greater inhibitory effect on ALL cell lines compared to primary fibroblasts. Flow cytometry and western blot analysis indicated that borrelidin was able to increase the level of apoptosis and cause G(1) arrest in ALL cell lines. Activation of the general control nonderepressible-2 (GCN2) kinase stress responsive pathway and induction of CHOP protein was significantly higher in ALL cell lines treated with borrelidin. These findings collectively suggest for the first time that borrelidin targets ALL cell lines by inducing apoptosis and mediating G(1) arrest and that borrelidin treatment in ALL cell lines is correlated with activation of the GCN2 kinase pathway.

Abstract 2642: Small molecule inhibitor borrelidin induces apoptosis in acute lymphoblastic leukemia cell lines in association with GCN2 kinase pathway activation

Abstract Introduction: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children. L-asparaginase (L-ASNase), an important agent in the treatment of ALL, catalyzes the hydrolysis of asparagine (Asn), leading to activation of the general control nonderepressible-2(GCN2) kinase stress-responsive pathway, inhibition of protein synthesis, and apoptotic cell death. Unfortunately, allergic hypersensitivity reactions can occur in patients after repeated administrations of ASNase and are the main reason for discontinuation of ASNase treatment. Borrelidin, a small molecule nitrile-containing macrolide, is a known inhibitor of bacterial and eukaryal threonyl-tRNA synthetase (ThrRS). As both ASNase and borrelidin increase the levels of uncharged tRNA, we hypothesize that ASNase may be replaced by borrelidin. Thus, the aim of the present study was to investigate if borrelidin could have a potential role in targeting ALL cell lines and, whether the GCN2 kinase pathway activation is involved as one of the downstream mechanisms by which borrelidin affects lymphoblastic cells. Methods: In order to mediate inhibition of threonyl-tRNA synthesis and to induce nutritional stress in ALL cell lines, Jurkat and CEM cells, cells were treated with borrelidin. Proliferation assay was also performed and the growth rate of these cells was studied. Propidium iodide staining and flow cytometry were performed to study the apoptotic effects of borrelidin on these cells as well as to study the effects of borrelidin on cell cycle progression. The levels of phosphorylated GCN2, total GCN2, phosphorylated eIF2α, total eIF2α, CHOP and cleaved PARP were evaluated by Western blot analysis. Results: Borrelidin was able to potently inhibit the proliferation and growth of ALL cell lines in low concentrations. Borrelidin showed a greater apoptotic effect on T cells compared to primary fibroblasts (half maximal inhibitory concentration (IC50) of 50 ng/ml and 375 ng/ml respectively). Flow cytometry and Western blot analysis indicated that borrelidin was able to increase the level of apoptosis in treated Jurkat cells as compared with non-treated control groups (3.9 ± 1.26 and 0.77 ± 0.06, respectively; p<0.01, n=3). Flow cytometry results showed a significant dose-dependent G1 arrest in Jurkat cells treated with borrelidin (10.88 ± 0.63) compared to that of non-treated control group (4.18 ± 0.24; p<0.001, n=3). Activation of the GCN2 kinase pathway as well as induction of the proapoptotic CHOP protein was significantly higher in ALL cell lines treated with borrelidin relative to the control non-treated cells. Conclusions: These findings collectively suggest for the first time that borrelidin may have promising potentials in treating ALL by activating the GCN2 kinase pathway, inducing apoptosis and mediating G1 arrest in T-cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2642. doi:10.1158/1538-7445.AM2011-2642

An unusual tRNAThr derived from tRNAHis reassigns in yeast mitochondria the CUN codons to threonine

The standard genetic code is used by most living organisms, yet deviations have been observed in many genomes, suggesting that the genetic code has been evolving. In certain yeast mitochondria, CUN codons are reassigned from leucine to threonine, which requires an unusual tRNAThr with an enlarged 8-nt anticodon loop (). To trace its evolutionary origin we performed a comprehensive phylogenetic analysis which revealed that evolved from yeast mitochondrial tRNAHis. To understand this tRNA identity change, we performed mutational and biochemical experiments. We show that Saccharomyces cerevisiae mitochondrial threonyl-tRNA synthetase (MST1) could attach threonine to both and the regular , but not to the wild-type tRNAHis. A loss of the first nucleotide (G−1) in tRNAHis converts it to a substrate for MST1 with a Km value (0.7 μM) comparable to that of (0.3 μM), and addition of G−1 to allows efficient histidylation by histidyl-tRNA synthetase. We also show that MST1 from Candida albicans, a yeast in which CUN codons remain assigned to leucine, could not threonylate , suggesting that MST1 has coevolved with . Our work provides the first clear example of a recent recoding event caused by alloacceptor tRNA gene recruitment.

Fidelity Escape by the Unnatural Amino Acid β-Hydroxynorvaline: An Efficient Substrate forEscherichia coliThreonyl-tRNA Synthetase with Toxic Effects on Growth

In all living systems, the fidelity of translation is maintained in part by the editing mechanisms of aminoacyl-tRNA synthetases (ARSs). Some nonproteogenic amino acids, including β-hydroxynorvaline (HNV) are nevertheless efficiently aminoacylated and become incorporated into proteins. To investigate the basis of HNV's ability to function in protein synthesis, the utilization of HNV by Escherichia coli threonyl-tRNA synthetase (ThrRS) was investigated through both in vitro functional experiments and bacterial growth studies. The measured specificity constant (k(cat)/K(M)) for HNV was found to be only 20-30-fold less than that of cognate threonine. The rate of aminoacyl transfer (10.4 s(-1)) was 10-fold higher than the multiple turnover k(cat) value (1 s(-1)), indicating that, as for cognate threonine, amino acid activation is likely to be the rate-limiting step. Like noncognate serine, HNV enhances the ATPase function of the synthetic site, at a rate not increased by nonaminoacylatable (3'-dA76) tRNA. ThrRS also failed to exhibit posttransfer editing activity against HNV. In growing bacteria, the addition of HNV dramatically suppressed growth rates, which indicates either negative phenotypic consequences associated with its incorporation into protein or inhibition of an unidentified metabolic reaction. The inability of wild ThrRS to prevent utilization of HNV as a substrate illustrates that, for at least one ARS, the naturally occurring enzyme lacks the capability to effectively discriminate against nonproteogenic amino acids that are not encountered under normal physiological conditions. Other examples of "fidelity escape" in the ARSs may serve as useful starting points in the design of ARSs with specificity for unnatural amino acids.

Biased gene transfer and its implications for the concept of lineage

BackgroundIn the presence of horizontal gene transfer (HGT), the concepts of lineage and genealogy in the microbial world become more ambiguous because chimeric genomes trace their ancestry from a myriad of sources, both living and extinct.ResultsWe present the evolutionary histories of three aminoacyl-tRNA synthetases (aaRS) to illustrate that the concept of organismal lineage in the prokaryotic world is defined by both vertical inheritance and reticulations due to HGT. The acquisition of a novel gene from a distantly related taxon can be considered as a shared derived character that demarcates a group of organisms, as in the case of the spirochaete Phenylalanyl-tRNA synthetase (PheRS). On the other hand, when organisms transfer genetic material with their close kin, the similarity and therefore relatedness observed among them is essentially shaped by gene transfer. Studying the distribution patterns of divergent genes with identical functions, referred to as homeoalleles, can reveal preferences for transfer partners. We describe the very ancient origin and the distribution of the archaeal homeoalleles for Threonyl-tRNA synthetases (ThrRS) and Seryl-tRNA synthetases (SerRS).ConclusionsPatterns created through biased HGT can be undistinguishable from those created through shared organismal ancestry. A re-evaluation of the definition of lineage is necessary to reflect genetic relatedness due to both HGT and vertical inheritance. In most instances, HGT bias will maintain and strengthen similarity within groups. Only in cases where HGT bias is due to other factors, such as shared ecological niche, do patterns emerge from gene phylogenies that are in conflict with those reflecting shared organismal ancestry.ReviewersThis article was reviewed by W. Ford Doolittle, François-Joseph Lapointe, and Frederic Bouchard.

Mechanistic insights into cognate substrate discrimination during proofreading in translation

Editing/proofreading by aminoacyl-tRNA synthetases is an important quality control step in the accurate translation of the genetic code that removes noncognate amino acids attached to tRNA. Defects in the process of editing result in disease conditions including neurodegeneration. While proofreading, the cognate amino acids larger by a methyl group are generally thought to be sterically rejected by the editing modules as envisaged by the "Double-Sieve Model." Strikingly using solution based direct binding studies, NMR-heteronuclear single quantum coherence (HSQC) and isothermal titration calorimetry experiments, with an editing domain of threonyl-tRNA synthetase, we show that the cognate substrate can gain access and bind to the editing pocket. High-resolution crystal structural analyses reveal that functional positioning of substrates rather than steric exclusion is the key for the mechanism of discrimination. A strategically positioned "catalytic water" molecule is excluded to avoid hydrolysis of the cognate substrate using a "RNA mediated substrate-assisted catalysis mechanism" at the editing site. The mechanistic proof of the critical role of RNA in proofreading activity is a completely unique solution to the problem of cognate-noncognate selection mechanism.

Aminoacyl Transfer Rate Dictates Choice of Editing Pathway in Threonyl-tRNA Synthetase

Aminoacyl-tRNA synthetases hydrolyze aminoacyl adenylates and aminoacyl-tRNAs formed from near-cognate amino acids, thereby increasing translational fidelity. The contributions of pre- and post-transfer editing pathways to the fidelity of Escherichia coli threonyl-tRNA synthetase (ThrRS) were investigated by rapid kinetics. In the pre-steady state, asymmetric activation of cognate threonine and noncognate serine was observed in the active sites of dimeric ThrRS, with similar rates of activation. In the absence of tRNA, seryl-adenylate was hydrolyzed 29-fold faster by the ThrRS catalytic domain than threonyl-adenylate. The rate of seryl transfer to cognate tRNA was only 2-fold slower than threonine. Experiments comparing the rate of ATP consumption to the rate of aminoacyl-tRNA(AA) formation demonstrated that pre-transfer hydrolysis contributes to proofreading only when the rate of transfer is slowed significantly. Thus, the relative contributions of pre- and post-transfer editing in ThrRS are subject to modulation by the rate of aminoacyl transfer.