Aminoacyl-tRNA synthetases database Y2K

The aminoacyl-tRNA synthetases (AARS) are a diverse group of enzymes that ensure the fidelity of transfer of genetic information from DNA into protein. They catalyse the attachment of amino acids to transfer RNAs and thereby establish the rules of the genetic code by virtue of matching the nucleotide triplet of the anticodon with its cognate amino acid. Currently, 818 AARS primary structures have been reported from archaebacteria, eubacteria, mitochondria, chloro-plasts and eukaryotic cells. The database is a compilation of the amino acid sequences of all AARSs, known to date, which are available as separate entries or alignments of related proteins via the WWW at http://rose.man.poznan.pl/aars/index.html     

Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process.

The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective. Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes. Nevertheless, the AARSs are very informative about the evolutionary process. Examination of the phylogenetic trees for each of the AARSs reveals the following. (i) Their evolutionary relationships mostly conform to established organismal phylogeny: a strong distinction exists between bacterial- and archaeal-type AARSs. (ii) Although the evolutionary profiles of the individual AARSs might be expected to be similar in general respects, they are not. It is argued that these differences in profiles reflect the stages in the evolutionary process when the taxonomic distributions of the individual AARSs became fixed, not the nature of the individual enzymes. (iii) Horizontal transfer of AARS genes between Bacteria and Archaea is asymmetric: transfer of archaeal AARSs to the Bacteria is more prevalent than the reverse, which is seen only for the "gemini group. " (iv) The most far-ranging transfers of AARS genes have tended to occur in the distant evolutionary past, before or during formation of the primary organismal domains. These findings are also used to refine the theory that at the evolutionary stage represented by the root of the universal phylogenetic tree, cells were far more primitive than their modern counterparts and thus exchanged genetic material in far less restricted ways, in effect evolving in a communal sense.     

Aminoacyl-tRNA Synthetases, the Genetic Code, and the Evolutionary Process

The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective. Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes. Nevertheless, the AARSs are very informative about the evolutionary process. Examination of the phylogenetic trees for each of the AARSs reveals the following. (i) Their evolutionary relationships mostly conform to established organismal phylogeny: a strong distinction exists between bacterial- and archaeal-type AARSs. (ii) Although the evolutionary profiles of the individual AARSs might be expected to be similar in general respects, they are not. It is argued that these differences in profiles reflect the stages in the evolutionary process when the taxonomic distributions of the individual AARSs became fixed, not the nature of the individual enzymes. (iii) Horizontal transfer of AARS genes between Bacteria and Archaea is asymmetric: transfer of archaeal AARSs to the Bacteria is more prevalent than the reverse, which is seen only for the “gemini group.” (iv) The most far-ranging transfers of AARS genes have tended to occur in the distant evolutionary past, before or during formation of the primary organismal domains. These findings are also used to refine the theory that at the evolutionary stage represented by the root of the universal phylogenetic tree, cells were far more primitive than their modern counterparts and thus exchanged genetic material in far less restricted ways, in effect evolving in a communal sense.     

氨酰-tRNA合成酶专一性识别的进化

氨酰-tRNA合成酶(AARS)是一类在蛋白质合成过程中起着重要作用的酶,它通过与tRNA及其相应氨基酸的专一性识别作用,使得基因序列能够被精确地翻译成蛋白质序列.然而,氨酰-tRNA合成酶的这种识别作用既有专一性,也具有“兼容性”.氨酰-tRNA合成酶的这种双重性质不仅与其结构的进化有关,而且还与其所处的各类生物的不同进化阶段有关.AARS似乎经历了一个由“模糊专一性”（多重专一性）到“精确专一性”（单一专一性）的演变历程.     

MIST,a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) constitute a family of RNA-binding proteins, that participate in the translation of the genetic code, by covalently linking amino acids to appropriate tRNAs. Due to their fundamental importance for cell life, AARSs are likely to be one of the most ancient families of enzymes and have therefore been characterized extensively. Paradoxically, little is known about their capacity to discriminate tRNAs mainly because of the practical challenges that represent precise and systematic tRNA identification. This work describes a new technical and conceptual approach named MIST (Microarray Identification of Shifted tRNAs) designed to study the formation of tRNA/AARS complexes independently from the aminoacylation reaction. MIST combines electrophoretic mobility shift assays with microarray analyses. Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition. In this study we focus on yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) and investigate in depth its ability to discriminate cellular tRNAs. We report that yArgRS in submicromolar concentrations binds cognate and non-cognate tRNAs with a wide range of apparent affinities. In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation. Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.     

Negative catalysis by the editing domain of class I aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (AARS) translate the genetic code by loading tRNAs with the cognate amino acids. The errors in amino acid recognition are cleared at the AARS editing domain through hydrolysis of misaminoacyl-tRNAs. This ensures faithful protein synthesis and cellular fitness. Using Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme, we demonstrated that the class I editing domain clears the non-cognate amino acids well-discriminated at the synthetic site with the same rates as the weakly-discriminated fidelity threats. This unveiled low selectivity suggests that evolutionary pressure to optimize the rates against the amino acids that jeopardize translational fidelity did not shape the editing site. Instead, we propose that editing was shaped to safeguard cognate aminoacyl-tRNAs against hydrolysis. Misediting is prevented by the residues that promote negative catalysis through destabilisation of the transition state comprising cognate amino acid. Such powerful design allows broad substrate acceptance of the editing domain along with its exquisite specificity in the cognate aminoacyl-tRNA rejection. Editing proceeds by direct substrate delivery to the editing domain (in cis pathway). However, we found that class I IleRS also releases misaminoacyl-tRNA^Ile and edits it in trans. This minor editing pathway was up to now recognized only for class II AARSs.     

Compositional dynamics and codon usage pattern of BRCA1 gene across nine mammalian species

The BRCA1 gene is located on the human chromosome 17q21.31 and plays important role in biological processes. The aminoacyl-tRNA synthetases (AARS) are a family of heterogenous enzymes responsible protein synthesis and whose secondary functions include a role in autoimmune myositis. Our findings reveal that the compositional constraint and the preference of more A/T –ending codons determine the codon usage patterns in BRCA1 gene while more G/C-ending codons influence the codon usage pattern of AARS gene among mammals. The codon usage bias in BRCA1 and AARS genes is low. The codon CGC encoding arginine amino acid and the codon TTA encoding leucine were uniformly distributed in BRCA1 and AARS genes, respectively in mammals including human. Natural selection might have played a major role while mutation pressure might have played a minor role in shaping the codon usage pattern of BRCA1 and AARS genes.     

Aminoacyl-tRNA synthetases database

Aminoacyl-tRNA synthetases (AARSs) are at the center of the question of the origin of life. They constitute a family of enzymes integrating the two levels of cellular organization: nucleic acids and proteins. AARSs arose early in evolution and are believed to be a group of ancient proteins. They are responsible for attaching amino acid residues to their cognate tRNA molecules, which is the first step in the protein synthesis. The role they play in a living cell is essential for the precise deciphering of the genetic code. The analysis of AARSs evolutionary history was not possible for a long time due to a lack of a sufficiently large number of their amino acid sequences. The emerging picture of synthetases' evolution is a result of recent achievements in genomics [Woese,C., Olsen,G.J., Ibba,M. and S?ll,D. (2000) Microbiol. Mol. Biol. Rev., 64, 202-236]. In this paper we present a short introduction to the AARSs database. The updated database contains 1047 AARS primary structures from archaebacteria, eubacteria, mitochondria, chloroplasts and eukaryotic cells. It is the compilation of amino acid sequences of all AARSs known to date, which are available as separate entries via the WWW at http://biobases.ibch.poznan.pl/aars/.     

The physiological target for LeuRS translational quality control is norvaline

The fidelity of protein synthesis depends on the capacity of aminoacyl‐tRNA synthetases (AARSs) to couple only cognate amino acid‐tRNA pairs. If amino acid selectivity is compromised, fidelity can be ensured by an inherent AARS editing activity that hydrolyses mischarged tRNAs. Here, we show that the editing activity of Escherichia coli leucyl‐tRNA synthetase (EcLeuRS) is not required to prevent incorrect isoleucine incorporation. Rather, as shown by kinetic, structural and in vivo approaches, the prime biological function of LeuRS editing is to prevent mis‐incorporation of the non‐standard amino acid norvaline. This conclusion follows from a reassessment of the discriminatory power of LeuRS against isoleucine and the demonstration that a LeuRS editing‐deficient E. coli strain grows normally in high concentrations of isoleucine but not under oxygen deprivation conditions when norvaline accumulates to substantial levels. Thus, AARS‐based translational quality control is a key feature for bacterial adaptive response to oxygen deprivation. The non‐essential role for editing under normal bacterial growth has important implications for the development of resistance to antimicrobial agents targeting the LeuRS editing site.     

Docking of tryptophanyl [corrected tryptophan] analogs to trytophanyl-tRNA synthetase: implications for non-canonical amino acid incorporations

Abstract Non-canonical amino acids (N(AA)), as building blocks for peptides and proteins during ribosomal translation, represent a nearly infinite supply of novel functions. The specific selection, activation and tRNA-charging of amino acids by aminoacyl-tRNA synthetases (AARS) in the aminoacylation reaction are essential steps. In most cases, aminoacylation of N(AA) is a good indication that the related amino acid will participate in ribosomal translation as well. However, testing the translational capacity of amino acid analogs has technical limitations. Therefore, a rapid and reliable in silico test for N(AA) recognition by AARS would be advantageous in experimental design. We chose tryptophanyl-tRNA synthetase from Escherichia coli as a model system for docking studies with various tryptophan analogs using the FlexX-Pharm strategy. We were able to calculate relative binding energies for Trp analogs in TrpRS that correlate well with their translational activities in E. coli. In particular, FlexX-Pharm predicted the binding sites of fluoro-, amino-, hydroxyl- and aza-containing Trp analogs within 1.5 A of Trp in the homology model of E. coli TrpRS. Therefore, the use of ligand docking prior to N(AA) incorporation experiments might provide a straightforward means for determining N(AA) that can be efficiently incorporated into a protein.     

Intraphylum diversity and complex evolution of cyanobacterial aminoacyl-tRNA synthetases

A comparative genomic analysis of 35 cyanobacterial strains has revealed that the gene complement of aminoacyl-tRNA synthetases (AARSs) and routes for aminoacyl-tRNA synthesis may differ among the species of this phylum. Several genes encoding AARS paralogues were identified in some genomes. In-depth phylogenetic analysis was done for each of these proteins to gain insight into their evolutionary history. GluRS, HisRS, ArgRS, ThrRS, CysRS, and Glu-Q-RS showed evidence of a complex evolutionary course as indicated by a number of inconsistencies with our reference tree for cyanobacterial phylogeny. In addition to sequence data, support for evolutionary hypotheses involving horizontal gene transfer or gene duplication events was obtained from other observations including biased sequence conservation, the presence of indels (insertions or deletions), or vestigial traces of ancestral redundant genes. We present evidences for a novel protein domain with two putative transmembrane helices recruited independently by distinct AARS in particular cyanobacteria.     

Aminoacyl-tRNA synthetases activity as a growth index in zooplankton

A simple method for the assay of aminoacyl-tRNA synthetases(AARS) activity was modified for application in planktonic crustaceansas an index of somatic growth. The cladoceran Daphnia magnawas cultured in the laboratory and its AARS activity measuredwithout substrate addition. The relationship between the enzymeactivities of animals of similar age and individual biomassgrowing at different rates was tested. A significant relationshipwas found between AARS activity and somatic growth in termsof both protein and dry weight.     

Assessment of Calanus finmarchicus growth and dormancy using the aminoacyl-tRNA synthetases method

We obtained growth rates of the copepod Calanus finmarchicusat different locations across the North Atlantic between May1998 and June 2004. Animals were incubated for 2–9 daysand fed either with natural food assemblages or with culturedalgae. During this period, we measured both somatic weight-specificgrowth rates (measured as protein change) and aminoacyl-tRNAsynthetases (AARS) activity. We found a highly significant relationshipbetween AARS activity and growth in protein content (R² = 0.55,P < 0.001). Significant AARS activity also occurred whengrowth was negative, the relationship predicting an AARS activitylevel of 8.33 nmPPi·mg protein^–1·h^–1when somatic growth is zero. This is because AARS activity isexpected even when growth is negative, owing to the continuedprotein turnover in the cells. The AARS method allowed for thefirst time the study of protein metabolism in overwinteringC. finmarchicus. Our study results showed that overwinteringcopepods had significantly lower values of AARS activity thannon-diapausing animals (t = –3.51, P < 0.002). TheAARS method opens the possibility to better understand physiologydynamics of deep-water organisms (e.g. the beginning and endof diapause).     

Evolution of acceptor stem tRNA recognition by class II prolyl-tRNA synthetase

Aminoacyl-tRNA synthetases (AARS) are an essential family of enzymes that catalyze the attachment of amino acids to specific tRNAs during translation. Previously, we showed that base-specific recognition of the tRNA(Pro) acceptor stem is critical for recognition by Escherichia coli prolyl-tRNA synthetase (ProRS), but not for human ProRS. To further delineate species-specific differences in acceptor stem recognition, atomic group mutagenesis was used to probe the role of sugar-phosphate backbone interactions in recognition of human tRNA(Pro). Incorporation of site-specific 2'-deoxynucleotides, as well as phosphorothioate and methylphosphonate modifications within the tRNA acceptor stem revealed an extensive network of interactions with specific functional groups proximal to the first base pair and the discriminator base. Backbone functional groups located at the base of the acceptor stem, especially the 2'-hydroxyl of A66, are also critical for aminoacylation catalytic efficiency by human ProRS. Therefore, in contrast to the bacterial system, backbone-specific interactions contribute significantly more to tRNA recognition by the human enzyme than base-specific interactions. Taken together with previous studies, these data show that ProRS-tRNA acceptor stem interactions have co-adapted through evolution from a mechanism involving 'direct readout' of nucleotide bases to one relying primarily on backbone-specific 'indirect readout'.     

Predicting sub-cellular localization of tRNA synthetases from their primary structures

Since endo-symbiotic events occur, all genes of mitochondrial aminoacyl tRNA synthetase (AARS) were lost or transferred from ancestral mitochondrial genome into the nucleus. The canonical pattern is that both cytosolic and mitochondrial AARSs coexist in the nuclear genome. In the present scenario all mitochondrial AARSs are nucleus-encoded, synthesized on cytosolic ribosomes and post-translationally imported from the cytosol into the mitochondria in eukaryotic cell. The site-based discrimination between similar types of enzymes is very challenging because they have almost same physico-chemical properties. It is very important to predict the sub-cellular location of AARSs, to understand the mitochondrial protein synthesis. We have analyzed and optimized the distinguishable patterns between cytosolic and mitochondrial AARSs. Firstly, support vector machines (SVM)-based modules have been developed using amino acid and dipeptide compositions and achieved Mathews correlation coefficient (MCC) of 0.82 and 0.73, respectively. Secondly, we have developed SVM modules using position-specific scoring matrix and achieved the maximum MCC of 0.78. Thirdly, we developed SVM modules using N-terminal, intermediate residues, C-terminal and split amino acid composition (SAAC) and achieved MCC of 0.82, 0.70, 0.39 and 0.86, respectively. Finally, a SVM module was developed using selected attributes of split amino acid composition (SA-SAAC) approach and achieved MCC of 0.92 with an accuracy of 96.00%. All modules were trained and tested on a non-redundant data set and evaluated using fivefold cross-validation technique. On the independent data sets, SA-SAAC based prediction model achieved MCC of 0.95 with an accuracy of 97.77%. The web-server 'MARSpred' based on above study is available at http://www.imtech.res.in/raghava/marspred/.     

Transfer RNA recognition by aminoacyl-tRNA synthetases

The aminoacyl-tRNA synthetases are an ancient group of enzymes that catalyze the covalent attachment of an amino acid to its cognate transfer RNA. The question of specificity, that is, how each synthetase selects the correct individual or isoacceptor set of tRNAs for each amino acid, has been referred to as the second genetic code. A wealth of structural, biochemical, and genetic data on this subject has accumulated over the past 40 years. Although there are now crystal structures of sixteen of the twenty synthetases from various species, there are only a few high resolution structures of synthetases complexed with cognate tRNAs. Here we review briefly the structural information available for synthetases, and focus on the structural features of tRNA that may be used for recognition. Finally, we explore in detail the insights into specific recognition gained from classical and atomic group mutagenesis experiments performed with tRNAs, tRNA fragments, and small RNAs mimicking portions of tRNAs.     

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity

Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or "charging") specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. This activity relieves ambiguities during translation of the genetic code that result from one synthetase activating multiple amino acid substrates. In this review, we describe methods that have been developed for assaying both pre- and post-transfer editing activities. Pre-transfer editing is defined as hydrolysis of a misactivated aminoacyl-adenylate prior to transfer to the tRNA. This reaction has been reported to occur either in the aminoacylation active site or in a separate editing domain. Post-transfer editing refers to the hydrolysis reaction that cleaves the aminoacyl-ester linkage formed between the carbonyl carbon of the amino acid and the 2' or 3' hydroxyl group of the ribose on the terminal adenosine. Post-transfer editing takes place in a hydrolytic active site that is distinct from the site of amino acid activation. Here, we focus on methods for determination of steady-state reaction rates using editing assays developed for both classes of synthetases.     

Recombinant DNA approaches for the development of metabolic systems used in in vitro toxicology.

In the past few years there has been considerable progress in the development of mammalian cell systems for use in genetic toxicology by the stable transfer of genes/cDNAs coding for drug metabolizing enzymes directly into the target cell. Alternative approaches have also been developed in which mammalian cells are transiently transfected with cDNAs coding for drug-metabolizing enzymes and S9 preparations expressing a single metabolizing enzyme isolated and used for metabolic activation. Progress in these areas is reviewed here and the relative merits of the different approaches are discussed. Work to date has focused primarily on the cytochrome P450 family of enzymes, although other enzyme systems involved in xenobiotic metabolism have been used. The central theme of this review is the transfer of genetic information to improve the metabolic capability of cell systems used in genetic toxicology. However, a basic philosophy of the review is that genetic manipulation of cultured mammalian cells has the potential for developing systems to be used to better understand chemically induced toxicological effects.     

New pathway of utilization of ammonia nitrogen for the synthesis of amino acids through NADH dependent transaminases in Bombyx mori L.

Abstract. NADH dependent transamination was recorded for the first time in silkworm Bombyx mori (L.) eggs and in larval tissues. L-Glutamine:2–oxoglutarate amino transferase (GOGAT) and L-asparagine:2–oxoglutarate amino transferase (AOGAT) were determined in embryonic and also in larval tissues of multi-and bivoltine races. The presence of these two enzymes coupled to glutamine synthetase indicated efficient utilization of metabolic ammonia hitherto unknown in higher organisms. It is proposed that these transfer enzymes help in building the free amino acid pool seen during the diapause and also in the fibroin synthesis in the last larval stadium.