Handling mammalian mitochondrial tRNAs and aminoacyl-tRNA synthetases for functional and structural characterization

The mammalian mitochondrial (mt) genome codes for only 13 proteins, which are essential components in the process of oxidative phosphorylation of ADP into ATP. Synthesis of these proteins relies on a proper mt translation machinery. While 22 tRNAs and 2 rRNAs are also coded by the mt genome, all other factors including the set of aminoacyl-tRNA synthetases (aaRSs) are encoded in the nucleus and imported. Investigation of mammalian mt aminoacylation systems (and mt translation in general) gains more and more interest not only in regard of evolutionary considerations but also with respect to the growing number of diseases linked to mutations in the genes of either mt-tRNAs, synthetases or other factors. Here we report on methodological approaches for biochemical, functional, and structural characterization of human/mammalian mt-tRNAs and aaRSs. Procedures for preparation of native and in vitro transcribed tRNAs are accompanied by recommendations for specific handling of tRNAs incline to structural instability and chemical fragility. Large-scale preparation of mg amounts of highly soluble recombinant synthetases is a prerequisite for structural investigations that requires particular optimizations. Successful examples leading to crystallization of four mt-aaRSs and high-resolution structures are recalled and limitations discussed. Finally, the need for and the state-of-the-art in setting up an in vitro mt translation system are emphasized. Biochemical characterization of a subset of mammalian aminoacylation systems has already revealed a number of unprecedented peculiarities of interest for the study of evolution and forensic research. Further efforts in this field will certainly be rewarded by many exciting discoveries.

Aminoacyl-tRNA synthetases: Probing the molecular basis of catalysis and discrimination

Aminoacyl-tRNA synthetases (RSs) are responsible for the essential connection of amino acids with trinucleotide sequences of tRNA's. The RS family constitutes two structurally dissimilar groups of proteins, class I and class II. Methionyl-tRNA synthetase (MetRS) and isoleucyl-tRNA synthetase (IleRS), both members of class I, were the focus of this work. Both enzymes are zinc-containing proteins; show a high degree of amino acid specificity; and edit activated noncognate amino acids, thereby ensuring the fidelity of the genetic code. The goals of this work were to further delineate the molecular basis of catalysis and discrimination in these enzymes by mapping active site geometries using high-resolution nuclear magnetic resonance spectroscopy (NMR).^ Internuclear distances obtained from transferred nuclear Overhauser effects were used to define the conformations of Mg($\alpha$,$\beta$-methylene)ATP bound to E. coli MetRS and E. coli IleRS in multiple complexes. Identical conformations were found for the bound ATP. Thus, the predicted structural homology between IleRS and MetRS is supported by consensus enzyme-bound nucleotide conformations. The conformation of the bound nucleotide is not sensitive to occupation of the amino acid site of MetRS or IleRS. Therefore, conformational changes known to occur in the synthetases upon ligand binding appear not to alter the bound conformation of the adenosine portion of the nucleotide. Nuclear Overhauser effects on the substrate amino acid L-selenomethionine were also used to evaluate the enzyme-bound conformation of the cognate amino acid. The amino acid assumes a conformation which is consistent with a proposed editing mechanism.^ The E. coli MetRS was shown to catalyze amino acid $\alpha$-proton exchange in the presence of deuterium oxide of all cognate amino acids. It is proposed that the enzyme-bound zinc coordinates the $\alpha$-carboxylate of the amino acid, rendering the $\alpha$-proton more acidic. An enzymic base is responsible for exchange of the $\alpha$-proton. This proposal suggests that the enzyme-bound zinc may have a role in amino acid discrimination in MetRS. However, the role of this exchange reaction in catalysis remains unknown. ^

The aminoacyl-tRNA synthetases of Drosophila melanogaster

Aminoacyl-tRNA synthetases (aaRSs) ligate amino acids to their cognate tRNAs, allowing them to decode the triplet code during translation. Through different mechanisms aaRSs also perform several non-canonical functions in transcription, translation, apoptosis, angiogenesis and inflammation. Drosophila has become a preferred system to model human diseases caused by mutations in aaRS genes, to dissect effects of reduced translation or non-canonical activities, and to study aminoacylation and translational fidelity. However, the lack of a systematic annotation of this gene family has hampered such studies. Here, we report the identification of the entire set of aaRS genes in the fly genome and we predict their roles based on experimental evidence and/or orthology. Further, we propose a new, systematic and logical nomenclature for aaRSs. We also review the research conducted on Drosophila aaRSs to date. Together, our work provides the foundation for further research in the fly aaRS field.

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 Söll,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://biobase s.ibch.poznan.pl/aars/.

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., 2000a) 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.

Translocation events in the evolution of aminoacyl-tRNA synthetases.

We have characterized hisS, the gene encoding the histidyl-tRNA synthetase (HisRS) from the tetraodontoid fish Fugu rubripes. The hisS gene is about 3.5 kbp long and contains 13 exons and 12 introns of 172 bp, on average. The Fugu hisS gene encodes a putative protein of 519 amino acids with the three motifs identified as signatures of class 2 aminoacyl-tRNA synthetases. A model for the shifting of intron 8 between Fugu and hamster is proposed based on the successive appearance of a cryptic splicing site followed by an insertion mutation that created a new acceptor site. In addition, sequence comparisons suggest that the hisS gene has undergone a translocation through the first intron. As a result, the Fugu HisRS has an N-terminal sequence markedly different from that in the human and hamster enzymes. We propose that similar events have been responsible for variations at the N-terminal end of other aminoacyl-tRNA synthetases. Our analysis suggests that this involves exchanges through introns of two exons encoding an ancestral 32-amino acid motif.

Sub-Cellular Localization and Complex Formation by Aminoacyl-tRNA Synthetases in Cyanobacteria: Evidence for Interaction of Membrane-Anchored ValRS with ATP Synthase

tRNAs are charged with cognate amino acids by aminoacyl-tRNA synthetases (aaRSs) and subsequently delivered to the ribosome to be used as substrates for gene translation. Whether aminoacyl-tRNAs are channeled to the ribosome by transit within translational complexes that avoid their diffusion in the cytoplasm is a matter of intense investigation in organisms of the three domains of life. In the cyanobacterium Anabaena sp. PCC 7120, the valyl-tRNA synthetase (ValRS) is anchored to thylakoid membranes by means of the CAAD domain. We have investigated whether in this organism ValRS could act as a hub for the nucleation of a translational complex by attracting other aaRSs to the membranes. Out of the 20 aaRSs, only ValRS was found to localize in thylakoid membranes whereas the other enzymes occupied the soluble portion of the cytoplasm. To investigate the basis for this asymmetric distribution of aaRSs, a global search for proteins interacting with the 20 aaRSs was conducted. The interaction between ValRS and the FoF1 ATP synthase complex here reported is of utmost interest and suggests a functional link between elements of the gene translation and energy production machineries.

Aminoacylation of tRNA in the evolution of an aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases catalyze aminoacylation of tRNAs by joining an amino acid to its cognate tRNA. The selection of the cognate tRNA is jointly determined by separate structural domains that examine different regions of the tRNA. The cysteine-tRNA synthetase of Escherichia coli has domains that select for tRNAs containing U73, the GCA anticodon, and a specific tertiary structure at the corner of the tRNA L shape. The E. coli enzyme does not efficiently recognize the yeast or human tRNACys, indicating the evolution of determinants for tRNA aminoacylation from E. coli to yeast to human and the coevolution of synthetase domains that interact with these determinants. By successively modifying the yeast and human tRNACys to ones that are efficiently aminoacylated by the E. coli enzyme, we have identified determinants of the tRNA that are important for aminoacylation but that have diverged in the course of evolution. These determinants provide clues to the divergence of synthetase domains. We propose that the domain for selecting U73 is conserved in evolution. In contrast, we propose that the domain for selecting the corner of the tRNA L shape diverged early, after the separation between E. coli and yeast, while that for selecting the GCA-containing anticodon loop diverged late, after the separation between yeast and human.

Twenty-first aminoacyl-tRNA synthetase–suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria

Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRS–suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli. The “21st synthetase–tRNA pairs” include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline tRNA. By using error-prone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can be stably expressed in E. coli. These mutants still aminoacylate the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E. coli proline tRNA by factors of 2.2- to 6.8-fold.

Interaction between human tRNA synthetases involves repeated sequence elements.

Aminoacyl-tRNA synthetases (tRNA synthetases) of higher eukaryotes form a multiprotein complex. Sequence elements that are responsible for the protein assembly were searched by using a yeast two-hybrid system. Human cytoplasmic isoleucyl-tRNA synthetase is a component of the multi-tRNA synthetase complex and it contains a unique C-terminal appendix. This part of the protein was used as bait to identify an interacting protein from a HeLa cDNA library. The selected sequence represented the internal 317 amino acids of human bifunctional (glutamyl- and prolyl-) tRNA synthetase, which is also known to be a component of the complex. Both the C-terminal appendix of the isoleucyl-tRNA synthetase and the internal region of bifunctional tRNA synthetase comprise repeating sequence units, two repeats of about 90 amino acids, and three repeats of 57 amino acids, respectively. Each repeated motif of the two proteins was responsible for the interaction, but the stronger interaction was shown by the native structures containing multiple motifs. Interestingly, the N-terminal extension of human glycyl-tRNA synthetase containing a single motif homologous to those in the bifunctional tRNA synthetase also interacted with the C-terminal motif of the isoleucyl-tRNA synthetase although the enzyme is not a component of the complex. The data indicate that the multiplicity of the binding motif in the tRNA synthetases is necessary for enhancing the interaction strength and may be one of the determining factors for the tRNA synthetases to be involved in the formation of the multi-tRNA synthetase complex.

Evolutionary History of Arabidopsis thaliana Aminoacyl-tRNA Synthetase Dual-Targeted Proteins

Aminoacyl-transfer RNA (tRNA) synthetases (aaRS) are key players in translation and act early in protein synthesis by mediating the attachment of amino acids to their cognate tRNA molecules. In plants, protein synthesis may occur in three subcellular compartments (cytosol, mitochondria, and chloroplasts), which requires multiple versions of the protein to be correctly delivered to its proper destination. The organellar aaRS are nuclear encoded and equipped with targeting information at the N-terminal sequence, which enables them to be specifically translocated to their final location. Most of the aaRS families present organellar proteins that are dual targeted to mitochondria and chloroplasts. Here, we examine the dual targeting behavior of aaRS from an evolutionary perspective. Our results show that Arabidopsis thaliana aaRS sequences are a result of a horizontal gene transfer event from bacteria. However, there is no evident bias indicating one single ancestor (Cyanobacteria or Proteobacteria). The dual-targeted aaRS phylogenetic relationship was characterized into two different categories (paralogs and homologs) depending on the state recovered for both dual-targeted and cytosolic proteins. Taken together, our results suggest that the dual-targeted condition is a gain-of-function derived from gene duplication. Selection may have maintained the original function in at least one of the copies as the additional copies diverged.

A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia

Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synthetase per amino acid, these organisms employ prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA formation. To date this dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) is only known to exist in archaea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA synthetase (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coli. By using G. lamblia, M. jannaschii, or E. coli tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro data were confirmed in vivo, as the cloned G. lamblia proS gene was able to complement a temperature-sensitive E. coli cysS strain. Inhibition studies of CysRS activity with proline analogs (thiaproline and 5′-O-[N-(l-prolyl)-sulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning and analysis of the corresponding cysS gene. Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified Saccharomyces cerevisiae and E. coli ProRS enzymes were unable to form Cys-tRNA under these conditions. Thus, the dual specificity is restricted to the archaeal genre of ProRS. G. lamblia's archaeal-type prolyl- and alanyl-tRNA synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems.

Wide cross-species aminoacyl-tRNA synthetase replacement in vivo: yeast cytoplasmic alanine enzyme replaced by human polymyositis serum antigen.

Because of variations in tRNA sequences in evolution, tRNA synthetases either do not acylate their cognate tRNAs from other organisms or execute misacylations which can be deleterious in vivo. We report here the cloning and primary sequence of a 958-aa Saccharomyces cerevisiae alanyl-tRNA synthetase. The enzyme is a close homologue of the human and Escherichia coli enzymes, particularly in the region of the primary structure needed for aminoacylation of RNA duplex substrates based on alanine tRNA acceptor stems with a G3.U70 base pair. An ala1 disrupted allele demonstrated that the gene is essential and that, therefore, ALA1 encodes an enzyme required for cytoplasmic protein synthesis. Growth of cells harboring the ala1 disrupted allele was restored by a cDNA clone encoding human alanyl-tRNA synthetase, which is a serum antigen for many polymyositis-afflicted individuals. The human enzyme in extracts from rescued yeast was detected with autoimmune antibodies from a polymyositis patient. We conclude that, in spite of substantial differences between human and yeast tRNA sequences in evolution, strong conservation of the G3.U70 system of recognition is sufficient to yield accurate aminoacylation in vivo across wide species distances.

Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications.

Universal trees based on sequences of single gene homologs cannot be rooted. Iwabe et al. [Iwabe, N., Kuma, K.-I., Hasegawa, M., Osawa, S. & Miyata, T. (1989) Proc. Natl. Acad. Sci. USA 86, 9355-9359] circumvented this problem by using ancient gene duplications that predated the last common ancestor of all living things. Their separate, reciprocally rooted gene trees for elongation factors and ATPase subunits showed Bacteria (eubacteria) as branching first from the universal tree with Archaea (archaebacteria) and Eucarya (eukaryotes) as sister groups. Given its topical importance to evolutionary biology and concerns about the appropriateness of the ATPase data set, an evaluation of the universal tree root using other ancient gene duplications is essential. In this study, we derive a rooting for the universal tree using aminoacyl-tRNA synthetase genes, an extensive multigene family whose divergence likely preceded that of prokaryotes and eukaryotes. An approximately 1600-bp conserved region was sequenced from the isoleucyl-tRNA synthetases of several species representing deep evolutionary branches of eukaryotes (Nosema locustae), Bacteria (Aquifex pyrophilus and Thermotoga maritima) and Archaea (Pyrococcus furiosus and Sulfolobus acidocaldarius). In addition, a new valyl-tRNA synthetase was characterized from the protist Trichomonas vaginalis. Different phylogenetic methods were used to generate trees of isoleucyl-tRNA synthetases rooted by valyl- and leucyl-tRNA synthetases. All isoleucyl-tRNA synthetase trees showed Archaea and Eucarya as sister groups, providing strong confirmation for the universal tree rooting reported by Iwabe et al. As well, there was strong support for the monophyly (sensu Hennig) of Archaea. The valyl-tRNA synthetase gene from Tr. vaginalis clustered with other eukaryotic ValRS genes, which may have been transferred from the mitochondrial genome to the nuclear genome, suggesting that this amitochondrial trichomonad once harbored an endosymbiotic bacterium.

Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference

The kinetoplast genetic code deviates from the universal code in that 90% of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA) is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA) anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs) have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi) to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite.