Paths of lateral gene transfer of lysyl-aminoacyl-tRNA synthetases with a unique evolutionary transition stage of prokaryotes coding for class I and II varieties by the same organisms

BACKGROUND: While the premise that lateral gene transfer (LGT) is a dominant evolutionary force is still in considerable dispute, the case for widespread LGT in the family of aminoacyl-tRNA synthetases (aaRS) is no longer contentious. aaRSs are ancient enzymes, guarding the fidelity of the genetic code. They are clustered in two structurally unrelated classes. Only lysine aminoacyl-tRNA synthetase (LysRS) is found both as a class 1 and a class 2 enzyme (LysRS1-2). Remarkably, in several extant prokaryotes both classes of the enzyme coexist, a unique phenomenon that has yet to receive its due attention. RESULTS: We applied a phylogenetic approach for determining the extent and origin of LGT in prokaryotic LysRS. Reconstructing species trees for Archaea and Bacteria, and inferring that their last common ancestors encoded LysRS1 and LysRS2, respectively, we studied the gains and losses of both classes. A complex pattern of LGT events emerged. In specific groups of organisms LysRS1 was replaced by LysRS2 (and vice versa). In one occasion, within the alpha proteobacteria, a LysRS2 to LysRS1 LGT was followed by reversal to LysRS2. After establishing the most likely LGT paths, we studied the possible origins of the laterally transferred genes. To this end, we reconstructed LysRS gene trees and evaluated the likely origins of the laterally transferred genes. While the sources of LysRS1 LGTs were readily identified, those for LysRS2 remain, for now, uncertain. The replacement of one LysRS by another apparently transits through a stage simultaneously coding for both synthetases, probably conferring a selective advantage to the affected organisms. CONCLUSION: The family of LysRSs features complex LGT events. The currently available data were sufficient for identifying unambiguously the origins of LysRS1 but not of LysRS2 gene transfers. A selective advantage is suggested to organisms encoding simultaneously LysRS1-2

Human tryptophanyl-tRNA synthetase is switched to a tRNA-dependent mode for tryptophan activation by mutations at V85 and I311

For most aminoacyl-tRNA synthetases (aaRS), their cognate tRNA is not obligatory to catalyze amino acid activation, with the exception of four class I (aaRS): arginyl-tRNA synthetase, glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase and class I lysyl-tRNA synthetase. Furthermore, for arginyl-, glutamyl- and glutaminyl-tRNA synthetase, the integrated 3' end of the tRNA is necessary to activate the ATP-PPi exchange reaction. Tryptophanyl-tRNA synthetase is a class I aaRS that catalyzes tryptophan activation in the absence of its cognate tRNA. Here we describe mutations located at the appended β1–β2 hairpin and the AIDQ sequence of human tryptophanyl-tRNA synthetase that switch this enzyme to a tRNA-dependent mode in the tryptophan activation step. For some mutant enzymes, ATP-PPi exchange activity was completely lacking in the absence of tRNATrp, which could be partially rescued by adding tRNATrp, even if it had been oxidized by sodium periodate. Therefore, these mutant enzymes have strong similarity to arginyl-tRNA synthetase, glutaminyl-tRNA synthetase and glutamyl-tRNA synthetase in their mode of amino acid activation. The results suggest that an aaRS that does not normally require tRNA for amino acid activation can be switched to a tRNA-dependent mode

Human tryptophanyl-tRNA synthetase is switched to a tRNA-dependent mode for tryptophan activation by mutations at V85 and I311

For most aminoacyl-tRNA synthetases (aaRS), their cognate tRNA is not obligatory to catalyze amino acid activation, with the exception of four class I (aaRS): arginyl-tRNA synthetase, glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase and class I lysyl-tRNA synthetase. Furthermore, for arginyl-, glutamyl- and glutaminyl-tRNA synthetase, the integrated 3' end of the tRNA is necessary to activate the ATP-PPi exchange reaction. Tryptophanyl-tRNA synthetase is a class I aaRS that catalyzes tryptophan activation in the absence of its cognate tRNA. Here we describe mutations located at the appended β1–β2 hairpin and the AIDQ sequence of human tryptophanyl-tRNA synthetase that switch this enzyme to a tRNA-dependent mode in the tryptophan activation step. For some mutant enzymes, ATP-PPi exchange activity was completely lacking in the absence of tRNATrp, which could be partially rescued by adding tRNATrp, even if it had been oxidized by sodium periodate. Therefore, these mutant enzymes have strong similarity to arginyl-tRNA synthetase, glutaminyl-tRNA synthetase and glutamyl-tRNA synthetase in their mode of amino acid activation. The results suggest that an aaRS that does not normally require tRNA for amino acid activation can be switched to a tRNA-dependent mode

The Complex Evolutionary History of Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) are a superfamily of enzymes responsible for the faithful translation of the genetic code and have lately become a prominent target for synthetic biologists. Our large-scale analysis of \u3e2500 prokaryotic genomes reveals the complex evolutionary history of these enzymes and their paralogs, in which horizontal gene transfer played an important role. These results show that a widespread belief in the evolutionary stability of this superfamily is misconceived. Although AlaRS, GlyRS, LeuRS, IleRS, ValRS are the most stable members of the family, GluRS, LysRS and CysRS often have paralogs, whereas AsnRS, GlnRS, PylRS and SepRS are often absent from many genomes. In the course of this analysis, highly conserved protein motifs and domains within each of the AARS loci were identified and used to build a web-based computational tool for the genome-wide detection of AARS coding sequences. This is based on hidden Markov models (HMMs) and is available together with a cognate database that may be used for specific analyses. The bioinformatics tools that we have developed may also help to identify new antibiotic agents and targets using these essential enzymes. These tools also may help to identify organisms with alternative pathways that are involved in maintaining the fidelity of the genetic code

Secondary Functions And Novel Inhibitors Of Aminoacyl-Trna Synthetases

The aminoacyl-tRNA synthetases are a family of enzymes involved in the process of translation, more specifically, ligating amino acids to their cognate tRNA molecules. Recent evidence suggests that aminoacyl-tRNA synthetases are capable of aminoacylating proteins, some of which are involved in the autophagy pathway. Here, we test the conditions under which E. coli and human threonyl-tRNA synthetases, as well as hisidyl-tRNA synthetase aminoacylate themselves. These reactions are ATP dependent, stimulated by Mg2+, and are inhibited by increasing cognate tRNA concentrations. These data represent the foundation for future aminoacylation experiments, specifically delving into the relationship between the autophagy pathway and the aminoacylation of proteins. Additionally, we provide evidence of the inhibitory abilities of the compound EHTS-0 on both E. coli and human threonyl-tRNA synthetases. Further, we also show that an EHTS-0 analog, EHTS-1, also significantly inhibits E. coli threonyl-tRNA synthetase but not the human enzyme. These data could be useful in determining the potential for EHTS-0 and EHTS-1 as possibly anti-angiogenic drugs

Macromolecular Databases – A Background of Bioinformatics

We propose a novel quasi‐Bayesian Metropolis‐within‐Gibbs algorithm that can be used to estimate drifts in the shock volatilities of a linearized dynamic stochastic general equilibrium (DSGE) model. The resulting volatility estimates differ from the existing approaches in two ways. First, the time variation enters non‐parametrically, so that our approach ensures consistent estimation in a wide class of processes, thereby eliminating the need to specify the volatility law of motion and alleviating the risk of invalid inference due to mis‐specification. Second, the conditional quasi‐posterior of the drifting volatilities is available in closed form, which makes inference straightforward and simplifies existing algorithms. We apply our estimation procedure to a standard DSGE model and find that the estimated volatility paths are smoother compared to alternative stochastic volatility estimates. Moreover, we demonstrate that our procedure can deliver statistically significant improvements to the density forecasts of the DSGE model compared to alternative methods.PostprintPeer reviewe

The complex evolutionary history of aminoacyl-tRNA synthetases

Σημείωση: διατίθεται συμπληρωματικό υλικό σε ξεχωριστό αρχείο

Changes In Threonyl-Trna Synthetase Expression And Secretion In Response To Endoplasmic Reticulum Stress By Monensin In Ovarian Cancer Cells

Aminoacyl-tRNA synthetases (ARS) are a family of enzymes that catalyze the charging of amino acids to their cognate tRNA in an aminoacylation reaction. Many members of this family have been found to have secondary functions independent of their primary aminoacylation function. Threonyl-tRNA synthetase (TARS), the ARS responsible for charging tRNA with threonine, is secreted from endothelial cells in response to both vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α), and stimulates angiogenesis and cell migration. Here we show a novel experimental approach for studying TARS secretion, and for observing the role of intracellular TARS in the endoplasmic reticulum (ER) stress response and in angiogenesis. Using Western blotting, immunofluorescence microscopy and RT-qPCR we were able to investigate changes in TARS protein and transcript levels. We initially hypothesized that TARS was secreted by exosomal release, and so we treated a human ovarian cancer cell line (CaOV-3) with monensin, an ionophore that increases exosome production, and VEGF to observe changes in intracellular and extracellular TARS protein. Monensin treatment consistently increased extracellular and intracellular TARS protein, however CD63, an exosome marker protein, levels were unaffected by monensin treatment. VEGF had no effect on intracellular TARS. We therefore hypothesized that the TARS response was a result of ER stress. The unfolded protein response (UPR) is a series of signaling pathways that are activated upon ER stress. When CaOV-3 cells were treated with increasing concentrations of monensin, intracellular levels of TARS and p-eIF2α, a downstream UPR target, increased accordingly. Monensin increased intracellular TARS protein and transcript levels in CaOV-3 cells. Monensin also increased DNAJB9, an ER chaperone protein, transcript levels, further confirming ER stress. Interestingly, monensin increased VEGF transcript levels about 6-fold. Borrelidin, a natural TARS inhibitor, also increased VEGF transcript levels, and caused an increase in p-eIF2α protein. Although the mechanism of TARS secretion remains unresolved, these data indicate that intracellular TARS expression increases in response to ER stress by monensin. Given TARS and VEGF transcript expression increased accordingly, it is possible that intracellular TARS may have pro-angiogenic function. Future directions may include investigating TARS interactions with translational control machinery

Zusammenspiel von posttranskriptionellen Modifikationen und Strukturen von humanen mitochondrialen tRNAs

Zusammenfassung Mitochondrien sind Organellen eukaryotischer Zellen, die für die Energieproduktion der Zellen verantwortlich sind, sowie über ein eigenes Genom verfügen. Das menschliche mitochondriale Genom kodiert 13 Proteine für die Untereinheiten der Atmungskettenkomplexe und 22 transfer RNAs (tRNAs). Mehr als 80 verschiedene Punktmutationen in den humanen mitochondrialen tRNA Genen werden mit Krankheiten wie Kardiopathie, Enzephalopathie und Myopathien assoziert. Die Bildung von alternativen sekundären und tertiären tRNA-Strukturen könnte die Pathogenität dieser Mutationen erklären. Um Informationen über die strukturelle Dynamik der humanen mitochondrialen tRNAs und über pathogene Mutanten (tRNALys-WT, -A8344G, tRNASer(UCN)-WT, -G7497A, -T7512C, tRNAGln-WT, -T4336C, tRNALeu(CUN)-WT, -A12320G, -G12315A) zu bekommen, wurde die Struktur von in vitro Transkripten mit folgenden Methoden genauer untersucht: chemische und enzymatische Kartierung, UV-Schmelzkurvenanalyse und elektrophoretische Mobilität auf nativen Gelen, sowie posttranskriptionelle Modifikation mit Enzymen aus humanen mitochondrialen Extrakten und aus Saccharomyces cerevisiae (scPus1p und scPus4p). Folgende Ergebnisse wurden dabei gewonnen: enzymatische und chemische Kartierungsdaten und UV-Schmelzkurvenanalyse zeigen, dass die A12320G und G12315A Mutanten der tRNALeu(CUN) eine ungewöhnliche Struktur besitzen, die sich vom Wildtyp deutlich unterscheidet. In humaner mitochondrialer tRNASer(UCN) führt die G7497A Mutation zu einer stark kompaktierten Struktur, was durch temperaturabhängige Strukturanalyse gezeigt wurde. Das durch diese Mutation eingeführte G•U Wobble-Paar im D-Stem hat einen negativen Einfluss auf die Erkennung sowohl durch humane mitochondriale Enzyme, als auch durch Pus4 und Pus1 aus S.cerevisiae. Die strukturelle Dynamik von nicht-modifizierten und teilmodifizierten Transkripten, die mit UV-Schmelzkurven analysiert wurden, zeigt, dass posttranskriptionelle Modifikationen eine wichtige Rolle in der strukturellen und wahrscheinlich auch in der metabolischen Stabilität dieser tRNA spielen. Die Studien der pathogenen G7497A Punktmutation in Osteosarcoma Zellen zeigen, dass untermodifizierte tRNAs instabil sind und in der Zelle schnell abgebaut werden. Es wurde eine Reduktion zur Verfügung stehender funktioneller tRNAs in Mitochondrien auf 10 % gefunden, was zu einer Verminderung der Proteinsynthese um 40 % führt [Mollers et al., 2005]. Insgesamt konnte in der vorliegenden Arbeit gezeigt werden, dass die meisten pathogenen Mutationen im Kernbereich der tRNA deren Struktur signifikant beeinflussen. Mit Ausnahme einer Mutation T4336C in tRNAGln, welche die Aktivität von Pseudouridin-Synthase im Vergleich zu dem Wildtyp erhöhte, zeigten die meisten pathogenen Mutationen eine Verringerung der Aktivität von den Modifikationsenzymen