Classes Of aminoacyl-tRNA Synthetases Research Articles

Amino acid activation analysis of primitive aminoacyl-tRNA synthetases encoded by both strands of a single gene using the malachite green assay

The Rodin-Ohno hypothesis postulates that two classes of aminoacyl-tRNA synthetases were encoded complementary to double-stranded DNA. Particularly, Geobacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS, belonging to class I) and Escherichia coli histidyl-tRNA synthetase (HisRS, belonging to class II) show high complementarity of the middle base of the codons in the mRNA sequence encoding each ATP binding site. Here, for the reported 46-residue peptides designed from the three-dimensional structures of TrpRS and HisRS, amino acid activation analysis was performed using the malachite green assay, which detects the pyrophosphate departing from ATP in the forward reaction of the first step of tRNA aminoacylation. A maltose-binding protein fusion with the 46 residues of TrpRS (TrpRS46mer) exhibited high activation capacity for several amino acids in the presence of ATP and amino acids, but the activity of an alanine substitution mutant of the first histidine in the HIGH motif (TrpRS46merH15A) was largely reduced. In contrast, pyrophosphate release by HisRS46mer in the histidine activation step was lower than that in the case of TrpRS46mer. Both HisRS46mer and the alanine mutant at the 113th arginine (HisRS46merR113A) showed slightly higher levels of pyrophosphate release than the maltose-binding protein alone. These results do not rule out the Rodin-Ohno hypothesis, but may suggest the necessity of establishing unique evolutionary models from different perspectives.

Combinatorial Fusion Rules to Describe Codon Assignment in the Standard Genetic Code

We propose combinatorial fusion rules that describe the codon assignment in the standard genetic code simply and uniformly for all canonical amino acids. These rules become obvious if the origin of the standard genetic code is considered as a result of a fusion of four protocodes: Two dominant AU and GC protocodes and two recessive AU and GC protocodes. The biochemical meaning of the fusion rules consists of retaining the complementarity between cognate codons of the small hydrophobic amino acids and large charged or polar amino acids within the protocodes. The proto tRNAs were assembled in form of two kissing hairpins with 9-base and 10-base loops in the case of dominant protocodes and two 9-base loops in the case of recessive protocodes. The fusion rules reveal the connection between the stop codons, the non-canonical amino acids, pyrrolysine and selenocysteine, and deviations in the translation of mitochondria. Using fusion rules, we predicted the existence of additional amino acids that are essential for the development of the standard genetic code. The validity of the proposed partition of the genetic code into dominant and recessive protocodes is considered referring to state-of-the-art hypotheses. The formation of two aminoacyl-tRNA synthetase classes is compatible with four-protocode partition.

The aminoacyl-tRNA synthetases had only a marginal role in the origin of the organization of the genetic code: Evidence in favor of the coevolution theory

The coevolution theory of the origin of the genetic code suggests that the organization of the genetic code coevolved with the biosynthetic relationships between amino acids. The mechanism that allowed this coevolution was based on tRNA-like molecules on which—this theory—would postulate the biosynthetic transformations between amino acids to have occurred. This mechanism makes a prediction on how the role conducted by the aminoacyl-tRNA synthetases (ARSs), in the origin of the genetic code, should have been. Indeed, if the biosynthetic transformations between amino acids occurred on tRNA-like molecules, then there was no need to link amino acids to these molecules because amino acids were already charged on tRNA-like molecules, as the coevolution theory suggests. In spite of the fact that ARSs make the genetic code responsible for the first interaction between a component of nucleic acids and that of proteins, for the coevolution theory the role of ARSs should have been entirely marginal in the genetic code origin. Therefore, I have conducted a further analysis of the distribution of the two classes of ARSs and of their subclasses—in the genetic code table—in order to perform a falsification test of the coevolution theory. Indeed, in the case in which the distribution of ARSs within the genetic code would have been highly significant, then the coevolution theory would be falsified since the mechanism on which it is based would not predict a fundamental role of ARSs in the origin of the genetic code. I found that the statistical significance of the distribution of the two classes of ARSs in the table of the genetic code is low or marginal, whereas that of the subclasses of ARSs statistically significant. However, this is in perfect agreement with the postulates of the coevolution theory. Indeed, the only case of statistical significance—regarding the classes of ARSs—is appreciable for the CAG code, whereas for its complement—the UNN/NUN code—only a marginal significance is measurable. These two codes codify roughly for the two ARS classes, in particular, the CAG code for the class II while the UNN/NUN code for the class I. Furthermore, the subclasses of ARSs show a statistical significance of their distribution in the genetic code table. Nevertheless, the more sensible explanation for these observations would be the following. The observation that would link the two classes of ARSs to the CAG and UNN/NUN codes, and the statistical significance of the distribution of the subclasses of ARSs in the genetic code table, would be only a secondary effect due to the highly significant distribution of the polarity of amino acids and their biosynthetic relationships in the genetic code. That is to say, the polarity of amino acids and their biosynthetic relationships would have conditioned the evolution of ARSs so that their presence in the genetic code would have been detectable. Even if the ARSs would not have—on their own—influenced directly the evolutionary organization of the genetic code. In other words, the role that ARSs had in the origin of the genetic code would have been entirely marginal. This conclusion would be in perfect accord with the predictions of the coevolution theory. Conversely, this conclusion would be in contrast—at least partially—with the physicochemical theories of the origin of the genetic code because they would foresee an absolutely more active role of ARSs in the origin of the organization of the genetic code.

A unified model of the standard genetic code.

The Rodin–Ohno (RO) and the Delarue models divide the table of the genetic code into two classes of aminoacyl-tRNA synthetases (aaRSs I and II) with recognition from the minor or major groove sides of the tRNA acceptor stem, respectively. These models are asymmetric but they are biologically meaningful. On the other hand, the standard genetic code (SGC) can be derived from the primeval RNY code (R stands for purines, Y for pyrimidines and N any of them). In this work, the RO-model is derived by means of group actions, namely, symmetries represented by automorphisms, assuming that the SGC originated from a primeval RNY code. It turns out that the RO-model is symmetric in a six-dimensional (6D) hypercube. Conversely, using the same automorphisms, we show that the RO-model can lead to the SGC. In addition, the asymmetric Delarue model becomes symmetric by means of quotient group operations. We formulate isometric functions that convert the class aaRS I into the class aaRS II and vice versa. We show that the four polar requirement categories display a symmetrical arrangement in our 6D hypercube. Altogether these results cannot be attained, neither in two nor in three dimensions. We discuss the present unified 6D algebraic model, which is compatible with both the SGC (based upon the primeval RNY code) and the RO-model.

Catalysis of tRNA Aminoacylation: Single Turnover to Steady-State Kinetics of tRNA Synthetases

Aminoacyl-tRNA synthetases (aaRS) catalyze the bimolecular association reaction between amino acid and tRNA by specifically and unerringly choosing the cognate amino acid and tRNA. There are two classes of such synthetases that perform tRNA-aminoacylation reaction. Interestingly, these two classes of aminoacyl-tRNA synthetases differ not only in their structures but they also exhibit remarkably distinct kinetics under pre-steady-state condition. The class I synthetases show initial burst of product formation followed by a slower steady-state rate. This has been argued to represent the influence of slow product release. In contrast, there is no burst in the case of class II enzymes. The tight binding of product with enzyme for class I enzymes is correlated with the enhancement of rate in presence of elongation factor EF-TU. In spite of extensive experimental studies, there is no detailed theoretical analysis that can provide a quantitative understanding of this important problem. In this article, we present a theoretical investigation of enzyme kinetics for both classes of aminoacyl-tRNA synthetases. We present an augmented kinetic scheme and then employ the methods of time-dependent probability statistics to obtain expressions for the first passage time distribution that gives both the time-dependent and the steady-state rates. The present study quantitatively explains all the above experimental observations. We propose an alternative path way in the case of class II enzymes showing the tRNA-dependent amino acid activation and the discrepancy between the single-turnover and steady-state rate.

Detection of Functional Overlapping Genes: Simulation and Case Studies

As far as protein-coding genes are concerned, there is a non-zero probability that at least one of the five possible overlapping sequences of any gene will contain an open-reading frame (ORF) of a length that may be suitable for coding a functional protein. It is, however, very difficult to determine whether or not such an ORF is functional. Recently, we proposed a method that predicts functionality of an overlapping ORF if it can be shown that it has been subject to purifying selection during its evolution. Here, we use simulation to test this method under several conditions and compare it with the method of Firth and Brown. We found that under most conditions, our method detects functional overlapping genes with higher sensitivity than Firth and Brown's method, while maintaining high specificity. Further, we tested the hypothesis that the two aminoacyl tRNA synthetase classes have originated from a pair of overlapping genes. A central piece of evidence ostensibly supporting this hypothesis is the assertion that an overlapping ORF of a heat-shock protein-70 gene, which exhibits some similarity to class 2 aminoacyl tRNA synthetases, is functional. We found signature of purifying selection only in highly divergent sequences, suggesting that the method yields false-positives in high sequence divergence and that the overlapping ORF is not a functional gene. Finally, we examined three cases of overlap in the human genome. We find varying signatures of purifying selection acting on these overlaps, raising the possibility that two of the overlapping genes may not be functional.

Control of Catalytic Cycle by a Pair of Analogous tRNA Modification Enzymes

Enzymes that use distinct active site structures to perform identical reactions are known as analogous enzymes. The isolation of analogous enzymes suggests the existence of multiple enzyme structural pathways that can catalyze the same chemical reaction. A fundamental question concerning analogous enzymes is whether their distinct active-site structures would confer the same or different kinetic constraints to the chemical reaction, particularly with respect to the control of enzyme turnover. Here, we address this question with the analogous enzymes of bacterial TrmD and its eukaryotic and archaeal counterpart Trm5. TrmD and Trm5 catalyze methyl transfer to synthesize the m1G37 base at the 3′ position adjacent to the tRNA anticodon, using S-adenosyl methionine (AdoMet) as the methyl donor. TrmD features a trefoil-knot active-site structure whereas Trm5 features the Rossmann fold. Pre-steady-state analysis revealed that product synthesis by TrmD proceeds linearly with time, whereas that by Trm5 exhibits a rapid burst followed by a slower and linear increase with time. The burst kinetics of Trm5 suggests that product release is the rate-limiting step of the catalytic cycle, consistent with the observation of higher enzyme affinity to the products of tRNA and AdoMet. In contrast, the lack of burst kinetics of TrmD suggests that its turnover is controlled by a step required for product synthesis. Although TrmD exists as a homodimer, it showed half-of-the-sites reactivity for tRNA binding and product synthesis. The kinetic differences between TrmD and Trm5 are parallel with those between the two classes of aminoacyl-tRNA synthetases, which use distinct active site structures to catalyze tRNA aminoacylation. This parallel suggests that the findings have a fundamental importance for enzymes that catalyze both methyl and aminoacyl transfer to tRNA in the decoding process.

Template-based structure prediction and molecular dynamics simulation study of two mammalian Aspartyl-tRNA synthetases

Aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of a specific amino acid. There are two classes of aminoacyl-tRNA synthetases. Class I usually exists as a monomeric or dimeric form and has two highly conserved sequence motifs. Functionally, it aminoacylates at the 2′-OH of an adenosine nucleotide. While, class II normally exists as a dimeric or tetrameric form and consists of three highly conserved sequence motifs. It aminoacylates at the 3′-OH of the same adenosine. Aspartyl-tRNA synthetase (AspRS) belongs to class II aaRSs, is not only important to sustain the mechanism of protein fidelity by specifically recognizing its cognate amino acid; but also equally significant in the aminoacylation of tRNA Asp. Several crystal structures of AspRS have been reported yet but no structural information is available for mammalian AspRS. In this study, we have applied template-based modeling/structure prediction to elucidate structural details of two mammalian AspRS from Homo sapiens and Mus musculus. The resultant models showed excellent stereochemistry similar to the crystal structure of yeast. A 5 ns molecular dynamics (MD) simulation was also performed to study the conformational changes occur in the flipping loop region (279–285). The root mean square fluctuation (RMSF) graph shows movements mostly in the catalytic site and in the flipping loop region while the main secondary structure maintained fairly stable conformations.

No Rosetta Stone for a Sense–Antisense Origin of Aminoacyl tRNA Synthetase Classes

Aminoacyl tRNA synthetases (aaRS) are crucial enzymes that join amino acids to their cognate tRNAs, thereby implementing the genetic code. These enzymes fall into two unrelated structural classes whose evolution has not been explained. The leading hypothesis, proposed by Rodin and Ohno, is that the two classes originated as a pair of sense-antisense genes encoded on opposite strands of a single DNA molecule. This unusual idea obtained its main support from reports of a "Rosetta stone": a locus where genes for heat shock protein 70 (HSP70) and an Nicotinamide adenine dinulecotide-specific glutamate dehydrogenase (NAD-GDH), which are structurally homologous to the two classes of aaRS, overlap extensively on complementary DNA strands. This remarkable locus was first characterized in the oomycete Achlya klebsiana and has since been reported in many other species. Here we present evidence that the open reading frames on the antisense strand of HSP70 genes are spurious, and we identify a more probable candidate for the gene encoding the oomycete NAD-GDH enzyme. These results cast extensive doubt on the Rosetta Stone argument.

On the origin of the genetic code: signatures of its primordial complementarity in tRNAs and aminoacyl-tRNA synthetases

If the table of the genetic code is rearranged to put complementary codons face-to-face, it becomes apparent that the code displays latent mirror symmetry with respect to two sterically different modes of tRNA recognition. These modes involve distinct classes of aminoacyl-tRNA synthetases (aaRSs I and II) with recognition from the minor or major groove sides of the acceptor stem, respectively. We analyze the anticodon pairs complementary to the face-to-face codon couplets. Taking into account the invariant nucleotides on either side (5' and 3'), we consider the risk of anticodon confusion and subsequent erroneous aminoacylation in the ancestral coding system. This logic leads to the conclusion that ribozymic precursors of tRNA synthetases had the same two complementary modes of tRNA aminoacylation. This surprising case of molecular mimicry (1) shows a key potential selective advantage arising from the partitioning of aaRSs into two classes, (2) is consistent with the hypothesis that the two aaRS classes were originally encoded by the complementary strands of the same primordial gene and (3) provides a 'missing link' between the classic genetic code, embodied in the anticodon, and the second, or RNA operational, code that is embodied mostly in the acceptor stem and is directly responsible for proper tRNA aminoacylation.

Aminoacyl-tRNA synthetase classes and groups in prokaryotes

Knowledge on the evolution of aminoacyl-tRNA synthetases is crucial to studies on the origins of life. The relationships between the different aminoacyl-tRNA synthetase specificities in prokaryotic organisms are studied in this work. We reconstructed the ancestor sequences and the phylogenetic relationships utilizing the Maximum Likelihood method. The results suggest that in class I the evolution of the N-terminal segment was strongly influenced by the amino acid hydropathy in both domains of prokaryotes. The results for the C-terminal segments of class I were different in the two domains, indicating that its evolution was strongly influenced by the specific types of tRNA modification in each domain. The class II groups in Archaea were more heterogeneous with respect to the hydropathy of amino acids, indicating the interference of other influences. In bacteria, the configuration was also complex but the overall consensual division in two groups was maintained, group IIa forming a single branch with the five hydroapathetic amino acid specificities and group IIb containing the specificities for the moderately hydrophobic together with the hydrophilic amino acids. It is indicated that the aminoacyl-tRNA synthetase in both domains were subjected to different selective forces in diverse parts of the proteins, resulting in complex phylogenetic patterns.

Distinct Kinetic Mechanisms of the Two Classes of Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases are divided into two classes based on both functional and structural criteria. Distinctions between the classes have heretofore been based on general features, such as the position of aminoacylation on the 3′-terminal tRNA ribose, and the topology and tRNA-binding orientation of the active-site protein fold. Here we show instead that transient burst kinetics provides a distinct mechanistic signature dividing the two classes of tRNA synthetases, and that this distinction has significant downstream effects on protein synthesis. Steady-state and transient kinetic analyses of class I CysRS and ValRS, and class II AlaRS and ProRS, reveal that class I tRNA synthetases are rate-limited by release of aminoacyl-tRNA, while class II synthetases are limited by a step prior to aminoacyl transfer. The tight aminoacyl-tRNA product binding by class I enzymes correlates with the ability of EF-Tu to form a ternary complex with class I but not class II synthetases, and the further capacity of this protein to enhance the rate of aminoacylation by class I synthetases. These results emphasize that the distinct mechanistic signatures of class I versus class II tRNA synthetases ensure rapid turnover of aminoacyl-tRNAs during protein synthesis.

Discrimination of cognate and noncognate substrates at the active site of class II lysyl-tRNA synthetase.

Within the two unrelated aminoacyl-tRNA synthetase classes, lysyl-tRNA synthetase (LysRS) is the only example known to exist in both classes. To probe the role of the amino acids responsible for L-lysine binding in the active site of the class II LysRS (LysRS2), we studied the lysS-encoded Escherichia coli protein. On the basis of the structure of L-lysine complexed with E. coli LysRS2 (lysS), residues implicated in amino acid recognition and discrimination were systematically replaced. Steady-state kinetic parameters for these variants showed reductions in the catalytic efficiency (k(cat)/K(M)) of 1-3 orders of magnitude, allowing the assignment of specific roles for key residues in the active site of LysRS2. To further investigate the role of each residue in discrimination against noncognate amino acids, steady-state kinetic parameters were determined for the nonprotein amino acid S-(2-aminoethyl)-L-cysteine, a potent inhibitor of LysRS2. While a number of variants showed reductions of several hundred-fold in efficiency of S-(2-aminoethyl)-L-cysteine utilization, this was uniformly accompanied by similar reductions in the efficiency of lysine utilization. Thus, manipulation of the amino acid binding site only allowed up to a 4-fold improvement in S-(2-aminoethyl)-L-cysteine discrimination. This is in contrast to the highly effective discrimination against S-(2-aminoethyl)-L-cysteine by class I LysRS and correlates with the fundamentally different roles of conserved aromatic residues in the two LysRS active sites. This now provides a mechanistic basis for the proposal that differences in amino acid discrimination have been pivotal in the evolution of two unrelated LysRSs.

On the classes of aminoacyl-tRNA synthetases, amino acids and the genetic code.

The division of the aminoacyl-tRNA synthetases in two classes is compared with a division of the amino acids in two classes, obtained from the AAIndex databank by a principal component analysis. The division of the enzymes in Classes I and II follows to a great extent a division in the chemical and biological properties of their cognate amino acids. Furthermore, the phylogenetic trees of Classes I and II enzymes are highly correlated with dendrograms obtained for their cognate amino acids by using the indices in the AAIndex database. We argue that the evolution of aminoacyl-tRNA synthetases was determined by the characteristics of their corresponding amino acids. We interpret these results considering models for the origin and evolution of the genetic code in which an initial version, containing fewer amino acids, was modified by the incorporation of new amino acids following duplication and divergence of previous synthetases and tRNA molecules.

On the 28-gon symmetry inherent in the genetic code intertwined with aminoacyl-tRNA synthetases—The Lucas series

Despite considerable efforts it has remained unclear what principle governs the selection of the 20 canonical amino acids in the genetic code. Based on a previous study of the 28-gonal and rotational symmetric arrangement of the 20 amino acids in the genetic code, new analyses of the organization of the genetic code system together with their intrinsic relation to the two classes of aminoacyl-tRNA synthetases are reported in this work. A close inspection revealed how the enzymes and the 20 gene-encoded amino acids are intertwined on the polyhedron model. Complementary and cooperative symmetries between class I and class II aminoacyl-tRNA synthetases displayed by a 28-gon organization are discussed, and we found that the two previously suggested evolutionary axes within the genetic code overlap the symmetry axes within the two classes of aminoacyl-tRNA synthetases. Moreover, it has been shown that the side-chain carbon-atom numbers (2, 1, 3, 4 and 7) in the overwhelming majority of the amino acids recognized by each of the two classes of aminoacyl-tRNA synthetases are determined by a mathematical relationship, the Lucas series. A stepwise co-evolutionary selection logic of the amino acids is manifested by the amino acid side-chain carbon-atom number balance at ‘17’, when grouping the genetic code doublets in the 28-gon organization. The number ‘17’ equals the sum of the initial five numbers in the Lucas series, which are 2, 1, 3, 4 and 7.

Equilibrium Unfolding of Bombyx mori Glycyl-tRNA Synthetase

Unfolding of Bombyx mori glycyl-tRNA synthetase was examined by multiple spectroscopic techniques. Tryptophan fluorescence of wild type enzyme and an N-terminally truncated form (N55) increased at low concentrations of urea or guanidine-HCl followed by a reduction in intensity at intermediate denaturant concentrations; a transition at higher denaturant was detected as decreased fluorescence intensity and a red-shifted emission. Solute quenching of fluorescence indicated that tryptophans become progressively solvent-exposed during unfolding. Wild type enzyme had stronger negative CD bands between 220 and 230 nm than the mutant, indicative of greater alpha-helical content. Urea or guanidine-HCl caused a reduction in ellipticity at 222 nm at low denaturant concentration with the wild type enzyme, a transition that is absent in the mutant; both enzymes exhibited a cooperative transition at higher denaturant concentrations. Both enzymes dissociate to monomers in 1.5 m urea. Unfolding of wild type enzyme is described by a multistate unfolding and a parallel two state unfolding; the two-state component is absent in the mutant. Changes in spectral properties associated with unfolding were largely reversible after dilution to low denaturant. Unfolding of glycyl-tRNA synthetase is complex with a native state, a native-like monomer, partially unfolded states, and the unfolded state.

On the Classes of Aminoacyl-tRNA Synthetases and the Error Minimization in the Genetic Code

As a consequence of the existence of two classes of aminoacyl-tRNA synthetases (aaRSs), we defined two types of mutations: g (mutations that do not change the class of the involved amino acids) and u (those which change the class). We have found that the mean chemical distance resulting from g mutations is smaller than that corresponding to u mutations, indicating that g mutations are responsible for most of the known minimization of the genetic code. This supports models for the origin and evolution of the code, in which new amino acids were added after duplications or modification of existing aaRSs.

A gene fusion event in the evolution of aminoacyl-tRNA synthetases

The genes of glutamyl- and prolyl-tRNA synthetases (GluRS and ProRS) are organized differently in the three kingdoms of the tree of life. In bacteria and archaea, distinct genes encode the two proteins. In several organisms from the eukaryotic phylum of coelomate metazoans, the two polypeptides are carried by a single polypeptide chain to form a bifunctional protein. The linker region is made of imperfectly repeated units also recovered as singular or plural elements connected as N-terminal or C-terminal polypeptide extensions in various eukaryotic aminoacyl-tRNA synthetases. Phylogenetic analysis points to the monophyletic origin of this polypeptide motif appended to six different members of the synthetase family, belonging to either of the two classes of aminoacyl-tRNA synthetases. In particular, the monospecific GluRS and ProRS from Caenorhabditis elegans, an acoelomate metazoan, exhibit this recurrent motif as a C-terminal or N-terminal appendage, respectively. Our analysis of the extant motifs suggests a possible series of events responsible for a gene fusion that gave rise to the bifunctional glutamyl-prolyl-tRNA synthetase through recombination between genomic sequences encoding the repeated units.

Protein evolution drives the evolution of the genetic code and vice versa

A model for the developmental pathway of the genetic code, grounded on group theory and the thermodynamics of codon–anticodon interaction is presented. At variance with previous models, it takes into account not only the optimization with respect to amino acid attributes but, also physicochemical constraints and initial conditions. A ‘simple-first’ rule is introduced after ranking the amino acids with respect to two current measures of chemical complexity. It is shown that a primeval code of only seven amino acids is enough to build functional proteins. It is assumed that these proteins drive the further expansion of the code. The proposed primeval code is compared with surrogate codes randomly generated and with another proposal for primeval code found in the literature. The departures from the ‘universal’ code, observed in many organisms and cellular compartments, fit naturally in the proposed evolutionary scheme. A strong correlation is found between, on one side, the two classes of aminoacyl-tRNA synthetases, and on the other, the amino acids grouped by end-atom-type and by codon type. An inverse of Davydov’s rules, to associate the amino acid end atoms (O/N and non-O/non-N) of 18 amino acids with codons containing a weak base (A/U), extended to the 20 amino acids, is derived.

The genetic code as a Golden mean determined system

For the first time it is shown that the genetic code, as a binary code, is determined by Golden mean through the unity of the binary-code tree and the Farey tree, corresponding to the Watson–Crick table and Gray code model of the genetic code at the same time. On the other hand, the Golden mean relations also correspond with the essentiality–nonessentiality of protein amino acids, as well as with their splitting into two classes handled by two classes of aminoacyl-tRNA synthetases.