On the evolution of structure in aminoacyl-tRNA synthetases.

The aminoacyl-tRNA synthetases are one of the major protein components in the translation machinery. These essential proteins are found in all forms of life and are responsible for charging their cognate tRNAs with the correct amino acid. The evolution of the tRNA synthetases is of fundamental importance with respect to the nature of the biological cell and the transition from an RNA world to the modern world dominated by protein-enzymes. We present a structure-based phylogeny of the aminoacyl-tRNA synthetases. By using structural alignments of all of the aminoacyl-tRNA synthetases of known structure in combination with a new measure of structural homology, we have reconstructed the evolutionary history of these proteins. In order to derive unbiased statistics from the structural alignments, we introduce a multidimensional QR factorization which produces a nonredundant set of structures. Since protein structure is more highly conserved than protein sequence, this study has allowed us to glimpse the evolution of protein structure that predates the root of the universal phylogenetic tree. The extensive sequence-based phylogenetic analysis of the tRNA synthetases (Woese et al., Microbiol. Mol. Biol. Rev. 64:202-236, 2000) has further enabled us to reconstruct the complete evolutionary profile of these proteins and to make connections between major evolutionary events and the resulting changes in protein shape. We also discuss the effect of functional specificity on protein shape over the complex evolutionary course of the tRNA synthetases.     

Nucleotide triplet based molecular phylogeny of class I and class II aminoacyl t-RNA synthetase in three domain of life process: bacteria, archaea, and eukarya

The aminoacyl-tRNA synthetases are one of the major protein components in the translation machinery. These essential proteins are found in all forms of life and are responsible for charging their cognate tRNAs with the correct amino acid. These important enzymes have been the subject of intense scientific inquiry for nearly half a century, but their complete evolutionary history has yet to emerge. Amino acids sequence based phylogeny has some limitation due to very low sequence similarity amongst the different tRNA synthetases and structure based phylogeny has also its limitation. In our study, tRNA nucleotide sequences of E. coli K12 (Bacteria), Saccharomyces cerevisiae (Eukarya), Thermococcus kodakaraensis KOD1, and Archaeoglobus fulgidus DSM 4304 (Archaea) were used for phylogenetic analysis. Our results complement the observation with the earlier studies based on multiple sequence alignment and structural alignment. We observed that relationship between archaeal tRNA synthetases are different that of bacteria and eucarya. Violation of Class rule of LysRS is observed here also. The uniqueness of this method is that it does not employ sequence alignment of complete nucleotide sequence of the corresponding gene.     

Structure-Based Phylogeny of Class IIa tRNA Synthetases in Relation to an Unusual Biochemistry

The available three-dimensional information for class II aminoacyl-tRNA synthetases has been used to generate sequence alignments that strictly adhere to the structural equivalencies between members of subclass IIa of these enzymes. The resulting alignments were used to study their phylogenetic relationships. In particular, the entire set of available sequences of prolyl-tRNA synthetases was analyzed in this way. In contrast to recent reports, we conclude that the evolutionary pattern of prolyl-tRNA synthetases does not obviously conform to the canonical phylogenetic distribution. The pattern found for these enzymes may be related to their biochemical characteristics. Our results indicate a potential relationship between the evolutionary pattern of prolyl-tRNA synthetases and the emergence of two enzymatically distinct forms of these proteins.     

Structural Phylogenomics Retrodicts the Origin of the Genetic Code and Uncovers the Evolutionary Impact of Protein Flexibility

The genetic code shapes the genetic repository. Its origin has puzzled molecular scientists for over half a century and remains a long-standing mystery. Here we show that the origin of the genetic code is tightly coupled to the history of aminoacyl-tRNA synthetase enzymes and their interactions with tRNA. A timeline of evolutionary appearance of protein domain families derived from a structural census in hundreds of genomes reveals the early emergence of the ‘operational’ RNA code and the late implementation of the standard genetic code. The emergence of codon specificities and amino acid charging involved tight coevolution of aminoacyl-tRNA synthetases and tRNA structures as well as episodes of structural recruitment. Remarkably, amino acid and dipeptide compositions of single-domain proteins appearing before the standard code suggest archaic synthetases with structures homologous to catalytic domains of tyrosyl-tRNA and seryl-tRNA synthetases were capable of peptide bond formation and aminoacylation. Results reveal that genetics arose through coevolutionary interactions between polypeptides and nucleic acid cofactors as an exacting mechanism that favored flexibility and folding of the emergent proteins. These enhancements of phenotypic robustness were likely internalized into the emerging genetic system with the early rise of modern protein structure.     

Structure,function and evolution of seryl-tRNA synthetases: Implications for the evolution of aminoacyl-tRNA synthetases and the genetic code

Two aspects of the evolution of aminoacyl-tRNA synthetases are discussed. Firstly, using recent crystal structure information on seryl-tRNA synthetase and its substrate complexes, the coevolution of the mode of recognition between seryl-tRNA synthetase and tRNA^ser in different organisms is reviewed. Secondly, using sequence alignments and phylogenetic trees, the early evolution of class 2 Amnoacyl-tRNA synthetases is traced. Arguments are presented to suggest that synthetases are not the oldest of protein enzymes, but survived as RNA enzymes during the early period of the evolution of protein catalysts. In this view, the relatedness of the current synthetases, as evidenced by the division into two classes with their associated subclasses, reflects the replacement of RNA synthetases by protein synthetases. This process would have been triggered by the acquisition of tRNA 3 end charging activity by early proteins capable of activating small molecules (e.g., amino acids) with ATP. If these arguments are correct, the genetic code was essentially frozen before the protein synthetases that we know today came into existence. Correspondence to: S. CusackBased on a presentation made at a workshop-Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code-held at Berkeley, CA, July 17–20, 1994     

Aminoacyl-tRNA synthetases

Crystallographic studies of a number of aminoacyl-tRNA synthetases and their complexes with ATP, amino acid and cognate tRNA are leading to an increasingly detailed picture of how these sophisticated enzymes function. Within the two distinct structural classes of ten synthetases, many common features are apparent, although evolution has led to many interesting idiosyncrasies in certain enzymes. Recent advances, specially concerning class II enzymes, have increased out knowledge of both the role of electrophiles in the mechanism of amino acid activation and cross-subunit tRNA recognition and help solve the evolutionary puzzles that have emerged from the extension of the aminoacyl-tRNA synthetase database to include Archae     

Basic faced alpha-helices are widespread in the peptide extensions of the eukaryotic aminoacyl-tRNA synthetases

Three aminoacyl-tRNA synthetases from yeast, one from plants and one from mammals possess unusual structures at their N termini, namely alpha helices with basic residues distributed asymmetrically, on a single face of the helix. It is unknown if these 'basic faced' alpha helices (BFAHs) are unique to the aminoacyl-tRNA synthetases. Analysis of the amino acid sequences of these five aminoacyl-tRNA synthetases using the hydrophobic moment algorithm failed to accurately identify the BFAHs. A new algorithm was therefore developed, called the 'basic moment'. This is a Fourier analysis procedure that predicts the distribution of basic residues within protein secondary structure. The basic moment identifies with a high degree of accuracy the five known BFAHs and also identifies further potential BFAHs at evolutionarily conserved positions in the peptide extensions of aspartyl-, lysyl- and valyl- tRNA synthetases from a range of eukaryotic species. In addition, the algorithm identifies the two-helix pair tRNA binding domain of alanyl-tRNA synthetase, implying that the domain includes a BFAH. The functional and evolutionary aspects of these structural features are discussed.     

Virus-encoded aminoacyl-tRNA synthetases: structural and functional characterization of mimivirus TyrRS and MetRS

Aminoacyl-tRNA synthetases are pivotal in determining how the genetic code is translated in amino acids and in providing the substrate for protein synthesis. As such, they fulfill a key role in a process universally conserved in all cellular organisms from their most complex to their most reduced parasitic forms. In contrast, even complex viruses were not found to encode much translation machinery, with the exception of isolated components such as tRNAs. In this context, the discovery of four aminoacyl-tRNA synthetases encoded in the genome of mimivirus together with a full set of translation initiation, elongation, and termination factors appeared to blur what was once a clear frontier between the cellular and viral world. Functional studies of two mimivirus tRNA synthetases confirmed the MetRS specificity for methionine and the TyrRS specificity for tyrosine and conformity with the identity rules for tRNA(Tyr) for archea/eukarya. The atomic structure of the mimivirus tyrosyl-tRNA synthetase in complex with tyrosinol exhibits the typical fold and active-site organization of archaeal-type TyrRS. However, the viral enzyme presents a unique dimeric conformation and significant differences in its anticodon binding site. The present work suggests that mimivirus aminoacyl-tRNA synthetases function as regular translation enzymes in infected amoebas. Their phylogenetic classification does not suggest that they have been acquired recently by horizontal gene transfer from a cellular host but rather militates in favor of an intricate evolutionary relationship between large DNA viruses and ancestral eukaryotes.     

Horizontal Gene Transfer in Aminoacyl-tRNA Synthetases Including Leucine-Specific Subtypes

Aminoacyl-tRNA synthetases catalyze a fundamental reaction for the flow of genetic information from RNA to protein. Their presence in all organisms known today highlights their important role in the early evolution of life. We investigated the evolutionary history of aminoacyl-tRNA synthetases on the basis of sequence data from more than 200 Archaea, Bacteria, and Eukaryota. Phylogenetic profiles are in agreement with previous observations that many genes for aminoacyl-tRNA synthetases were transferred horizontally between species from all domains of life. We extended these findings by a detailed analysis of the history of leucyl-tRNA synthetases. Thereby, we identified a previously undetected case of horizontal gene transfer from Bacteria to Archaea based on phylogenetic profiles, trees, and networks. This means that, finally, the last subfamily of aminoacyl-tRNA synthetases has lost its exceptional position as the sole subfamily that is devoid of horizontal gene transfer. Furthermore, the leucyl-tRNA synthetase phylogenetic tree suggests a dichotomy of the archaeal/eukaryotic-cytosolic and bacterial/eukaryotic-mitochondrial proteins. We argue that the traditional division of life into Prokaryota (non-chimeric) and Eukaryota (chimeric) is favorable compared to Woese’s trichotomy into Archaea/Bacteria/Eukaryota. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Yves Van de Peer]     

Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases

Class 2 aminoacyl-tRNA synthetases, which include the enzymes for alanine, aspartic acid, asparagine, glycine, histidine, lysine, phenylalanine, proline, serine and threonine, are characterised by three distinct sequence motifs 1,2 and 3 (reference 1). The structural and evolutionary relatedness of these ten enzymes are examined using alignments of primary sequences from prokaryotic and eukaryotic sources and the known three dimensional structure of seryl-tRNA synthetase from E. coli. It is shown that motif 1 forms part of the dimer interface of seryl-tRNA synthetase and motifs 2 and 3 part of the putative active site. It is further shown that the seven alpha 2 dimeric synthetases can be subdivided into class 2a (proline, threonine, histidine and serine) and class 2b (aspartic acid, asparagine and lysine), each subclass sharing several important characteristic sequence motifs in addition to those characteristic of class 2 enzymes in general. The alpha 2 beta 2 tetrameric enzymes (for glycine and phenylalanine) show certain special features in common as well as some of the class 2b motifs. In the alanyl-tRNA synthetase only motif 3 and possibly motif 2 can be identified. The sequence alignments suggest that the catalytic domain of other class 2 synthetases should resemble the antiparallel domain found in seryl-tRNA synthetase. Predictions are made about the sequence location of certain important helices and beta-strands in this domain as well as suggestions concerning which residues are important in ATP and amino acid binding. Strong homologies are found in the N-terminal extensions of class 2b synthetases and in the C-terminal extensions of class 2a synthetases suggesting that these putative tRNA binding domains have been added at a later stage in evolution to the catalytic domain.     

Codases: 50 years after

Aminoacyl-tRNA synthetases (codases) catalyze aminoacylation of a particular tRNA with the corresponding amino acid at the first step of protein biosynthesis. The review considers the universal structural and functional characteristics of this largest family of enzymes, partitioned into two classes. The modes of tRNA binding and recognition, as well as additional editing activity, which are responsible for the extremely high fidelity of aminoacyl-tRNA synthesis, are discussed. The available data suggest an unusual evolutionary history for the most important components of the mechanism that ensures the proper synthesis of proteins and the association of this mechanism with amino acid biosynthesis. In addition, the review considers the secondary functions of synthetases in various cell metabolic processes, including pathophysiological ones. Their investigation may help to develop new diagnostic techniques and therapies.     

Functional expansion of human tRNA synthetases achieved by structural inventions

Known as an essential component of the translational apparatus, the aminoacyl-tRNA synthetase family catalyzes the first step reaction in protein synthesis, that is, to specifically attach each amino acid to its cognate tRNA. While preserving this essential role, tRNA synthetases developed other roles during evolution. Human tRNA synthetases, in particular, have diverse functions in different pathways involving angiogenesis, inflammation and apoptosis. The functional diversity is further illustrated in the association with various diseases through genetic mutations that do not affect aminoacylation or protein synthesis. Here we review the accumulated knowledge on how human tRNA synthetases used structural inventions to achieve functional expansions.     

Adaptation to tRNA acceptor stem structure by flexible adjustment in the catalytic domain of class I tRNA synthetases

Class I aminoacyl-tRNA synthetases (aaRSs) use a Rossmann-fold domain to catalyze the synthesis of aminoacyl-tRNAs required for decoding genetic information. While the Rossmann-fold domain is conserved in evolution, the acceptor stem near the aminoacylation site varies among tRNA substrates, raising the question of how the conserved protein fold adapts to RNA sequence variations. Of interest is the existence of an unpaired C-A mismatch at the 1-72 position unique to bacterial initiator tRNA(fMet) and absent from elongator tRNAs. Here we show that the class I methionyl-tRNA synthetase (MetRS) of Escherichia coli and its close structural homolog cysteinyl-tRNA synthetase (CysRS) display distinct patterns of recognition of the 1-72 base pair. While the structural homology of the two enzymes in the Rossmann-fold domain is manifested in a common burst feature of aminoacylation kinetics, CysRS discriminates against unpaired 1-72, whereas MetRS lacks such discrimination. A structure-based alignment of the Rossmann fold identifies the insertion of an α-helical motif, specific to CysRS but absent from MetRS, which docks on 1-72 and may discriminate against mismatches. Indeed, substitutions of the CysRS helical motif abolish the discrimination against unpaired 1-72. Additional structural alignments reveal that with the exception of MetRS, class I tRNA synthetases contain a structural motif that docks on 1-72. This work demonstrates that by flexible insertion of a structural motif to dock on 1-72, the catalytic domain of class I tRNA synthetases can acquire structural plasticity to adapt to changes at the end of the tRNA acceptor stem.     

Monophyly of class I aminoacyl tRNA synthetase,USPA, ETFP,photolyase, and PP-ATPase nucleotide-binding domains: implications for protein evolution in the RNA

Protein sequence and structure comparisons show that the catalytic domains of Class I aminoacyl-tRNA synthetases, a related family of nucleotidyltransferases involved primarily in coenzyme biosynthesis, nucleotide-binding domains related to the UspA protein (USPA domains), photolyases, electron transport flavoproteins, and PP-loop-containing ATPases together comprise a distinct class of alpha/beta domains designated the HUP domain after HIGH-signature proteins, UspA, and PP-ATPase. Several lines of evidence are presented to support the monophyly of the HUP domains, to the exclusion of other three-layered alpha/beta folds with the generic "Rossmann-like" topology. Cladistic analysis, with patterns of structural and sequence similarity used as discrete characters, identified three major evolutionary lineages within the HUP domain class: the PP-ATPases; the HIGH superfamily, which includes class I aaRS and related nucleotidyltransferases containing the HIGH signature in their nucleotide-binding loop; and a previously unrecognized USPA-like group, which includes USPA domains, electron transport flavoproteins, and photolyases. Examination of the patterns of phyletic distribution of distinct families within these three major lineages suggests that the Last Universal Common Ancestor of all modern life forms encoded 15-18 distinct alpha/beta ATPases and nucleotide-binding proteins of the HUP class. This points to an extensive radiation of HUP domains before the last universal common ancestor (LUCA), during which the multiple class I aminoacyl-tRNA synthetases emerged only at a late stage. Thus, substantial evolutionary diversification of protein domains occurred well before the modern version of the protein-dependent translation machinery was established, i.e., still in the RNA world.     

Genomics and the evolution of aminoacyl-tRNA synthesis.

Translation is the process by which ribosomes direct protein synthesis using the genetic information contained in messenger RNA (mRNA). Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. Thus, the sequential pairing of codons in mRNA with tRNA anticodons determines the order of amino acids in a protein. It is therefore imperative for accurate translation that tRNAs are only coupled to amino acids corresponding to the RNA anticodon. This is mostly, but not exclusively, achieved by the direct attachment of the appropriate amino acid to the 3'-end of the corresponding tRNA by the aminoacyl-tRNA synthetases. To ensure the accurate translation of genetic information, the aminoacyl-tRNA synthetases must display an extremely high level of substrate specificity. Despite this highly conserved function, recent studies arising from the analysis of whole genomes have shown a significant degree of evolutionary diversity in aminoacyl-tRNA synthesis. For example, non-canonical routes have been identified for the synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Characterization of non-canonical aminoacyl-tRNA synthesis has revealed an unexpected level of evolutionary divergence and has also provided new insights into the possible precursors of contemporary aminoacyl-tRNA synthetases.     

Recent advances in RNA-protein interaction studies

Conclusions The RNA binding sites for several small proteins have been characterised. These sites include double helical regions with hairpins, bulged bases and internal loops. As seen in Flock House virus structure, some proteins may recognise phosphate backbone of the canonical A-form helix not in a sequence-specific manner. If sequence-specific base contacts are to be made, then the A-helic major groove must be widened. This can be accomplished by introducing bulges, internal loops and hairpin loops into double helical regions. In these cases proteins may recognise both distorted backbone conformations and read out base sequences in a widened major groove. Crystallographic studies on complexes of aminoacyl-tRNA synthetase and tRNA showed that even RNAs with stable tertiary fold undergo substantial structural changes upon binding to the synthetases. The structural variability of RNA as well as the ability of RNA to distort upon protein binding may be crucial in RNA-protein interactions.     

Evolution of tRNA recognition systems and tRNA gene sequences

The aminoacylation of tRNAs by the aminoacyl-tRNA synthetases recapitulates the genetic code by dictating the association between amino acids and tRNA anticodons. The sequences of tRNAs were analyzed to investigate the nature of primordial recognition systems and to make inferences about the evolution of tRNA gene sequences and the evolution of the genetic code. Evidence is presented that primordial synthetases recognized acceptor stem nucleotides prior to the establishment of the three major phylogenetic lineages. However, acceptor stem sequences probably did not achieve a level of sequence diversity sufficient to faithfully specify the anticodon assignments of all 20 amino acids. This putative bottleneck in the evolution of the genetic code may have been alleviated by the advent of anticodon recognition. A phylogenetic analysis of tRNA gene sequences from the deep Archaea revealed groups that are united by sequence motifs which are located within a region of the tRNA that is involved in determining its tertiary structure. An association between the third anticodon nucleotide (N36) and these sequence motifs suggests that a tRNA-like structure existed close to the time that amino acid-anticodon assignments were being established. The sequence analysis also revealed that tRNA genes may evolve by anticodon mutations that recruit tRNAs from one isoaccepting group to another. Thus tRNA gene evolution may not always be monophyletic with respect to each isoaccepting group.Based on a presentation made at a workshop— Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code—held at Berkeley, CA, July 17–20, 1994 Correspondence to: M.E. Saks     

Role of nuclear pools of aminoacyl-tRNA synthetases in tRNA nuclear export

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 20001) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export.     

Small-angle X-ray Solution Scattering Study of the Multi-aminoacyl-tRNA Synthetase Complex Reveals an Elongated and Multi-armed particle

In animal cells, nine aminoacyl-tRNA synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-aminoacyl-tRNA synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex.