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     

Origin and evolution of the mitochondrial aminoacyl-tRNA synthetases

Many theories favor a fusion of 2 prokaryotic genomes for the origin of the Eukaryotes, but there are disagreements on the origin, timing, and cellular structures of the cells involved. Equally controversial is the source of the nuclear genes for mitochondrial proteins, although the alpha-proteobacterial contribution to the mitochondrial genome is well established. Phylogenetic inferences show that the nuclearly encoded mitochondrial aminoacyl-tRNA synthetases (aaRSs) occupy a position in the tree that is not close to any of the currently sequenced alpha-proteobacterial genomes, despite cohesive and remarkably well-resolved alpha-proteobacterial clades in 12 of the 20 trees. Two or more alpha-proteobacterial clusters were observed in 8 cases, indicative of differential loss of paralogous genes or horizontal gene transfer. Replacement and retargeting events within the nuclear genomes of the Eukaryotes was indicated in 10 trees, 4 of which also show split alpha-proteobacterial groups. A majority of the mitochondrial aaRSs originate from within the bacterial domain, but none specifically from the alpha-Proteobacteria. For some aaRS, the endosymbiotic origin may have been erased by ongoing gene replacements on the bacterial as well as the eukaryotic side. For others that accurately resolve the alpha-proteobacterial divergence patterns, the lack of affiliation with mitochondria is more surprising. We hypothesize that the ancestral eukaryotic gene pool hosted primordial "bacterial-like" genes, to which a limited set of alpha-proteobacterial genes, mostly coding for components of the respiratory chain complexes, were added and selectively maintained.     

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.     

Molecular evolution: aminoacyl-tRNA synthetases on the loose

Modified versions - paralogs - of the catalytic domain of at least three different aminoacyl-tRNA synthetases have been found to serve catalytic or regulatory roles in other reactions. These findings suggest that the first modern tRNA-synthetases could have been derived from amino-acid biosynthetic enzymes.     

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.     

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.     

One plasmid selection system for the rapid evolution of aminoacyl-tRNA synthetases

We have developed a rapid, straightforward, one plasmid dual positive/negative selection system for the evolution of aminoacyl-tRNA synthetases with altered specificities in Escherichia coli. This system utilizes an amber stop codon containing chloramphenicol acetyltransferase/uracil phosphoribosyltransferase fusion gene. We demonstrate the utility of the system by identifying a variant of the Methanococcus jannaschii tyrosyl synthetase from a library of 10⁹ variants that selectively incorporates para-iodophenylalanine in response to an amber stop codon.     

Speculations on the evolution of the genetic code IV the evolution of the aminoacyl-tRNA synthetases

An evolutionary scheme is postulated in which a primitive code, involving only guanine and cytosine, would code for glycine(GG.), alanine(GC), arginine(CG.) and proline(CC). There evolves from this primitive code families of related amino acids as the code expands. The evolution of the aminiacyl-tRNA synthetases are considered to be indicators for the evolution of the genetic code. The postulated model for the evolution of the genetic code is used to give an evolutionary interpretation to the recent work on the structure and sequences of the aminoacyl-tRNA synthetases.     

Peculiarities of aminoacyl-tRNA synthetases from trypanosomatids

Trypanosomatid parasites are responsible for various human diseases, such as sleeping sickness, animal trypanosomiasis, or cutaneous and visceral leishmaniases. The few available drugs to fight related parasitic infections are often toxic and present poor efficiency and specificity, and thus, finding new molecular targets is imperative. Aminoacyl-tRNA synthetases (aaRSs) are essential components of the translational machinery as they catalyze the specific attachment of an amino acid onto cognate tRNA(s). In trypanosomatids, one gene encodes both cytosolic- and mitochondrial-targeted aaRSs, with only three exceptions. We identify here a unique specific feature of aaRSs from trypanosomatids, which is that most of them harbor distinct insertion and/or extension sequences. Among the 26 identified aaRSs in the trypanosome Leishmania tarentolae, 14 contain an additional domain or a terminal extension, confirmed in mature mRNAs by direct cDNA nanopore sequencing. Moreover, these RNA-Seq data led us to address the question of aaRS dual localization and to determine splice-site locations and the 5′-UTR lengths for each mature aaRS-encoding mRNA. Altogether, our results provided evidence for at least one specific mechanism responsible for mitochondrial addressing of some L. tarentolae aaRSs. We propose that these newly identified features of trypanosomatid aaRSs could be developed as relevant drug targets to combat the diseases caused by these parasites.     

Multienzyme complexes of eukaryotic aminoacyl-tRNA synthetases

Eukaryotic aminoacyl-tRNA synthetases, unlike their prokaryotic counterparts, may occur as high-Mr multienzyme complexes. Recently, successful purification of synthetase complexes makes possible the elucidation of the structural organization of these high-Mr complexes. Although their physiological significance remains unknown, recent studies suggest some possible functional roles for these complexes.     

Common peptides study of aminoacyl-tRNA synthetases

Background

Aminoacyl tRNA synthetases (aaRSs) constitute an essential enzyme super-family, providing fidelity of the translation process of mRNA to proteins in living cells. They are common to all kingdoms and are of utmost importance to all organisms. It is thus of great interest to understand the evolutionary relationships among them and underline signature motifs defining their common domains.

Results

We utilized the Common Peptides (CPs) framework, based on extracted deterministic motifs from all aaRSs, to study family-specific properties. We identified novel aaRS–class related signatures that may supplement the current classification methods and provide a basis for identifying functional regions specific to each aaRS class. We exploited the space spanned by the CPs in order to identify similarities between aaRS families that are not observed using sequence alignment methods, identifying different inter-aaRS associations across different kingdom of life. We explored the evolutionary history of the aaRS families and evolutionary origins of the mitochondrial aaRSs. Lastly, we showed that prevalent CPs significantly overlap known catalytic and binding sites, suggesting that they have meaningful functional roles, as well as identifying a motif shared between aaRSs and a the Biotin-[acetyl-CoA carboxylase] synthetase (birA) enzyme overlapping binding sites in both families.

Conclusions

The study presents the multitude of ways to exploit the CP framework in order to extract meaningful patterns from the aaRS super-family. Specific CPs, discovered in this study, may play important roles in the functionality of these enzymes. We explored the evolutionary patterns in each aaRS family and tracked remote evolutionary links between these families.     

Evolution of genes,evolution of species: the case of aminoacyl-tRNA synthetases

All of the aminoacyl-tRNA synthetase (aaRS) sequences currently available in the data banks have been subjected to a systematic analysis aimed at finding gene duplications, genetic recombinations, and horizontal transfers. Evidence is provided for the occurrence (or probable occurrence) of such phenomena within this class of enzymes. In particular, it is suggested that the monomeric PheRS from the yeast mitochondrion is a chimera of the alpha and beta chains of the standard tetrameric protein. In addition, it is proposed that the dimeric and tetrameric forms of GlyRS are the result of a double and independent acquisition of the same specificity within two different subclasses of aaRS. The phylogenetic reconstructions of the evolutionary histories of the genes encoding aaRS are shown to be extremely diverse. While large segments of the population are consistent with the broad grouping into the three Woesian domains, some phylogenetic reconstructions do not place the Archae and the Eucarya as sister groups but, rather, show a gram-negative bacteria/eukaryote clustering. In addition, many individual genes pose difficulties that preclude any simple evolutionary scheme. Thus, aaRS's are clearly a paradigm of F. Jacob's "odd jobs of evolution" but, on the whole, do not call into question the evolutionary scenario originally proposed by Woese and subsequently refined by others.     

The new aspects of aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (AARS) are essential proteins found in all living organisms. They form a diverse group of enzymes that ensure the fidelity of transfer of genetic information from the DNA into the protein. AARS 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. Here we summarise the effects of recent studies on this interesting family of multifunctional enzymes.     

Evolutionary anomalies among the aminoacyl-tRNA synthetases

Unexpected relationships among the various aminoacyl-tRNA synthetases continue to be uncovered. The question arises — is this mainly the result of promiscuous exchange, or is the confusion really a reflection of the differential loss of past duplications? Phylogenetic analysis may yet provide the answer.     

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.