Charged tRNA Research Articles

Quality control in tRNA charging -- editing of homocysteine.

All living organisms conduct protein synthesis with a high degree of accuracy maintained in the transmission and flow of information from a gene to protein product. One crucial 'quality control' point in maintaining a high level of accuracy is the selectivity by which aminoacyl-tRNA synthetases furnish correctly activated amino acids, attached to tRNA species, as the building blocks for growing protein chains. When differences in binding energies of amino acids to an aminoacyl-tRNA synthetase are inadequate, editing is used as a major determinant of enzyme selectivity. Some incorrect amino acids are edited at the active site before the transfer to tRNA (pre-transfer editing), while others are edited after transfer to tRNA at a separate editing site (post-transfer editing). Access of natural non-protein amino acids, such as homocysteine, homoserine, or ornithine to the genetic code is prevented by the editing function of aminoacyl-tRNA synthetases. Disabling editing function leads to tRNA mischarging errors and incorporation of incorrect amino acids into protein, which is detrimental to cell homeostasis and inhibits growth. Continuous homocysteine editing by methionyl-tRNA synthetase, resulting in the synthesis of homocysteine thiolactone, is part of the process of tRNA aminoacylation in living organisms, from bacteria to man. Excessive homocysteine thiolactone synthesis in hyperhomocysteinemia caused by genetic or nutritional deficiencies is linked to human vascular and neurological diseases.

The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A

The highly conserved Kinase, Endopeptidase and Other Proteins of small Size (KEOPS)/Endopeptidase-like and Kinase associated to transcribed Chromatin (EKC) protein complex has been implicated in transcription, telomere maintenance and chromosome segregation, but its exact function remains unknown. The complex consists of five proteins, Kinase-Associated Endopeptidase (Kae1), a highly conserved protein present in bacteria, archaea and eukaryotes, a kinase (Bud32) and three additional small polypeptides. We showed that the complex is required for a universal tRNA modification, threonyl carbamoyl adenosine (t6A), found in all tRNAs that pair with ANN codons in mRNA. We also showed that the bacterial ortholog of Kae1, YgjD, is required for t6A modification of Escherichia coli tRNAs. The ATPase activity of Kae1 and the kinase activity of Bud32 are required for the modification. The yeast protein Sua5 has been reported previously to be required for t6A synthesis. Using yeast extracts, we established an in vitro system for the synthesis of t6A that requires Sua5, Kae1, threonine, bicarbonate and ATP. It remains to be determined whether all reported defects of KEOPS/EKC mutants can be attributed to the lack of t6A, or whether the complex has multiple functions.

Expression, purification and ligand binding properties of the recombinant translation initiation factor (PeIF5B) from Pisum sativum.

Gene encoding a novel translation initiation factor PeIF5B from Pisum sativum with sequence similarity to eIF5B from H. sapiens, D. melanogaster, S. cerevisiae as well as archaeal aIF5B from M. thermoautotrophicum was earlier reported by us. We now describe the expression and purification of 96 kDa recombinant PeIF5B (rPeIF5B) protein. Using fluorescence and circular dichroism spectra analyses, we show that Mg(2+) binding does not lead to any change in PeIF5B aromatic amino acid micro-environment, whereas GTP binding induces significant changes in the local environment of the aromatic amino acids. However, the protein undergoes changes in secondary structure upon metal ion and nucleotide binding. Charged initiator tRNA binding to PeIF5B is found to be cofactor dependent. PeIF5B binds to GTP in vitro as evident from autoradiography. Based on homology modeling of the catalytic domain of PeIF5B, we could confirm the conformational changes in PeIF5B following ligand binding.

Frameshift Mutation of MARS Gene Encoding an Aminoacyl-tRNA Synthetase in Gastric and Colorectal Carcinomas with Microsatellite Instability

To the editor, Gene translation is one of the most fundamental biologic processes for cells. Aminoacyl-tRNA synthetases (ARSs) are enzymes that charge tRNAs with their cognate amino acids during the translation.1 Of the ARSs, nine ARSs form macromolecular complexes with ARS-interacting multifunctional proteins (AIMP1, 2 and 3). Many of the complex-forming ARSs and AIMPs play roles not only in gene translation, but also in other biological processes such as apoptosis, angiogenesis and inflammation.1 It has been known that AIMP genes may act as tumor suppressor genes.2 Although non-translational roles of the complex-forming ARSs are known, their roles in cancer biology remain unknown. Microsatellite instability (MSI) is defined by length alterations in repeated DNA sequences, and 10-30% of colorectal cancer (CRC) and gastric cancer (GC) are classified as MSI-positive cancers.3 Many tumor suppressor genes in cancers with MSI harbor frameshift mutations at the repeats in coding sequences.3 By analyzing public database, we found a T9 repeat in exon 3 of methionyl-tRNA synthetase (MARS) gene (nucleotides 204-212) that had not been analyzed for the mutations in cancers. To see whether the T9 is mutated in GC and CRC with MSI, we analyzed them by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP). We analyzed 30 high-MSI (MSI-H) and 15 low-MSI (MSI-L) GC, and 40 MSI-H and nine MSI-L CRC. Malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle by microdissection as described previously.4 Tumor and normal DNA were amplified with by PCR using a primer pair that could amplify the T9 in exon 3 (product size 118 bps). Radioisotope ([32P]dCTP) was incorporated into the PCR for detection by SSCP. After SSCP, mobility shifts compared to wild-type bands were analyzed by visual inspection. Direct DNA sequencing was performed in the cancers with mobility shifts in the SSCP. We repeated the experiments twice to ensure the specificity of the results. PCR-SSCP analysis identified aberrant bands in two of the GC with MSI-H (2/30; 6.7%) and one of the CRC with MSI-H (1/40; 2.5%), but not in those with MSI-L. DNA from normal tissue showed no shifts in SSCP, indicating the mutations had risen somatically. Direct DNA sequencing of the cancers with the aberrant bands led to identification of a recurrent MARS mutation in the T9 repeat sequences (Fig. 1). The mutation was c.212delT, which would result in premature stops of the amino acid synthesis (p.Leu71CysfsX33). Fig. 1 Frameshift mutation of MARS in a gastric carcinoma with MSI. Direct DNA sequencing analysis of MARS exon 3 from a gastric adenocarcinoma with MSI-H shows a heterozygous T deletion in tumor tissue as compare to normal tissue. Human MARS exists as a component of the multi-ARS macromolecular complex, but this protein can dissociate from the complex and move to nucleus.5 After entering the nucleus, MARS plays a role in biogenesis of rRNA in nucleoli.5 Human MARS (about 900 amino acids long) contains N-terminal 267 amino acids and C-terminal 40 amino acids that are involved in protein-protein interactions. The intervening sequences are for its catalytic functions. Also, there are two nuclear localization signals (NLSs) (amino acids 897-900 and 724-730).5 The frameshift mutation of MARS identified in this study would lead to premature stops of amino acid syntheses in MARS protein (p.Leu71CysfsX33) and would abolish most of the domains of MARS (the catalytic domain, the NLSs and parts of the protein interaction domains). The MARS mutation may alter the structure of MARS, and might inactivate the functions of MARS. However, the sequencing data of the mutation (heterozygous mutation) suggest that the second allele may be intact. It is possible that the catalytic function of MARS for the protein translation that is essential for viability of cells may be preserved by the intact second allele. The mutant allele may alter the non-catalytic functions of MARS and might play a role in cancer pathogenesis. However, to date, the background data on non-catalytic functions of MARS is not enough to interpret the role of the mutant MARS in cancers. In this study, we for the first time identified an ARS-encoding gene mutation in human cancers. To our knowledge, there has been no report on functions of MARS protein related to MSI. To address consequences of such down-regulations in cancer development (especially related to cancers with MSI-H), functional studies on the mutated genes and their products should be further performed. Also, to see whether mutation of MARS gene is a common feature of cancers, the gene status should be further analyzed in other cancers.

Structure of a tryptophanyl-tRNA synthetase containing an iron-sulfur cluster.

A novel aminoacyl-tRNA synthetase that contains an iron-sulfur cluster in the tRNA anticodon-binding region and efficiently charges tRNA with tryptophan has been found in Thermotoga maritima. The crystal structure of TmTrpRS (tryptophanyl-tRNA synthetase; TrpRS; EC 6.1.1.2) reveals an iron-sulfur [4Fe-4S] cluster bound to the tRNA anticodon-binding (TAB) domain and an L-tryptophan ligand in the active site. None of the other T. maritima aminoacyl-tRNA synthetases (AARSs) contain this [4Fe-4S] cluster-binding motif (C-x₂₂-C-x₆-C-x₂-C). It is speculated that the iron-sulfur cluster contributes to the stability of TmTrpRS and could play a role in the recognition of the anticodon.

Methionine Deprivation Regulates the S6K1 Pathway and Protein Synthesis in Avian QM7 Myoblasts without Activating the GCN2/eIF2 alpha Cascade1–3

Amino acids modulate mRNA translation through the 70 kDa ribosomal protein S6 kinase (S6K1) and the general control nondepressible 2 protein kinase (GCN2)/eukaryotic initiation factor 2 alpha eIF2 alpha pathways. The aim of the present study was therefore to explore the signaling cascades potentially modulated by methionine availability in quail muscle QM7 myoblasts using media providing all other amino acids. Methionine deprivation caused a lower S6K1 phosphorylation compared with control (Ctl) cells. Supplying the methionine-deprived media with L- and DL-methionine isomers restored S6K1 phosphorylation to the levels observed in Ctl cells. Methionine also regulated downstream S6K1 targets (i.e. ribosomal protein S6 and eukaryotic elongation factor 2), modulated translation preinitiation complex (PIC) assembly, and stimulated protein synthesis. Replacing the lacking methionine with D-methionine or its hydroxyanalog [2-hydroxy-(4-methylthio) butanoic acid] did not restore S6K1 activation or protein synthesis. Conversely, the S6K1 pathway was activated by a methionine precursor, the ketoanalog of methionine. Methionine availability regulated the GCN2/eIF2 alpha pathway. However, our results indicate that methionine deprivation led to lower protein synthesis without activating eIF2 alpha phosphorylation, a process known to limit the formation of the 43S PIC. Using the amino acid alcohol methioninol did not decrease S6K1 phosphorylation or activity and did not alter the regulation of protein synthesis by methionine. These findings suggest that methionine exerts an effect on S6K1 signaling and protein synthesis in avian QM7 myoblasts through a mechanism partly independent of the global regulation via tRNA charging.

Selective control of amino acid metabolism by the GCN2 eIF2 kinase pathway in Saccharomyces cerevisiae

BackgroundWhen eukaryotic cells are deprived of amino acids, uncharged tRNAs accumulate and activate the conserved GCN2 protein kinase. Activated Gcn2p up-regulates the general amino acid control pathway through phosphorylation of the translational initiation factor eIF2. In Saccharomyces cerevisiae, Gcn2p is the only kinase that phosphorylates eIF2 to regulate translation through this mechanism. We addressed changes in yeast growth and tRNA aminoacylation, or charging, during amino acid depletion in the presence and absence of GCN2. tRNA charging was measured using a microarray technique which simultaneously measures all cytosolic tRNAs. A fully prototrophic strain, and its isogenic gcn2Δ counterpart, were used to study depletion for each of the 20 amino acids, with a focus on Trp, Arg, His and Leu, which are metabolically distinct and together provide a good overview on amino acid metabolism.ResultsWhile the wild-type strain had no observable phenotype upon depletion for any amino acid, the gcn2Δ strain showed slow growth in media devoid of only Trp or Arg. Consistent with the growth phenotypes, profiles of genome-wide tRNA charging revealed significant decrease in cognate tRNA charging only in the gcn2Δ strain upon depletion for Trp or Arg. In contrast, there was no change in tRNA charging during His and Leu depletion in either the wild-type or gcn2Δ strains, consistent with the null effect on growth during loss of these amino acids. We determined that the growth phenotype of Trp depletion is derived from feedback inhibition of aromatic amino acid biosynthesis. By removing Phe and Tyr from the media in addition to Trp, regular growth was restored and tRNATrp charging no longer decreased. The growth phenotype of Arg depletion is derived from unbalanced nitrogen metabolism. By supplementing ornithine upon Arg depletion, both growth and tRNAArg charging were partially restored.ConclusionUnder mild stress conditions the basal activity of Gcn2p is sufficient to allow for proper adaptation to amino acid depletion. This study highlights the importance of the GCN2 eIF2 kinase pathway for maintaining metabolic homeostasis, contributing to appropriate tRNA charging and growth adaptation in response to culture conditions deficient for the central amino acids, tryptophan and arginine.

The DamX protein of Escherichia coli and Salmonella enterica

We recently showed that disruption of damX causes bile sensitivity in Salmonella enterica. The damX gene is part of an operon that contains genes with heterogeneous functions: DNA adenine methylation, biosynthesis of aromatic compounds, carbohydrate metabolism, and tRNA charging. The damX gene encodes a protein with a predicted size of 46 kDa. In Salmonella, DamX is found in the inner membrane of both dividing and non-dividing cells. The DamX protein contains a peptidoglycan-binding SPOR domain, and accumulates in the E. coli septal ring. E. coli mutants lacking DamX are bile-sensitive like their Salmonella counterparts.

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Functional Characterization of Alternate Optimal Solutions of Escherichia coli's Transcriptional and Translational Machinery

The constraint-based reconstruction and analysis approach has recently been extended to describe Escherichia coli's transcriptional and translational machinery. Here, we introduce the concept of reaction coupling to represent the dependency between protein synthesis and utilization. These coupling constraints lead to a significant contraction of the feasible set of steady-state fluxes. The subset of alternate optimal solutions (AOS) consistent with maximal ribosome production was calculated. The majority of transcriptional and translational reactions were active for all of these AOS, showing that the network has a low degree of redundancy. Furthermore, all calculated AOS contained the qualitative expression of at least 92% of the known essential genes. Principal component analysis of AOS demonstrated that energy currencies (ATP, GTP, and phosphate) dominate the network's capability to produce ribosomes. Additionally, we identified regulatory control points of the network, which include the transcription reactions of σ70 (RpoD) as well as that of a degradosome component (Rne) and of tRNA charging (ValS). These reactions contribute significant variance among AOS. These results show that constraint-based modeling can be applied to gain insight into the systemic properties of E. coli's transcriptional and translational machinery.

Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNAMet formylation in Apicomplexa

Apicomplexans possess three translationally active compartments: the cytosol, a single tubular mitochondrion, and a vestigial plastid organelle called apicoplast. Mitochondrion and apicoplast are of bacterial evolutionary origin and therefore depend on a bacterial-like translation machinery. The minimal mitochondrial genome contains only three ORFs, and in Toxoplasma gondii the absence of mitochondrial tRNA genes is compensated for by the import of cytosolic eukaryotic tRNAs. Although all compartments require a complete set of charged tRNAs, the apicomplexan nuclear genomes do not hold sufficient aminoacyl-tRNA synthetase (aaRSs) genes to be targeted individually to each compartment. This study reveals that aaRSs are either cytosolic, apicoplastic or shared between the two compartments by dual targeting but are absent from the mitochondrion. Consequently, tRNAs are very likely imported in their aminoacylated form. Furthermore, the unexpected absence of tRNA(Met) formyltransferase and peptide deformylase implies that the requirement for a specialized formylmethionyl-tRNA(Met) for translation initiation is bypassed in the mitochondrion of Apicomplexa.

Atlas of rice grain filling-related metabolism under high temperature: joint analysis of metabolome and transcriptome demonstrated inhibition of starch accumulation and induction of amino acid accumulation.

High temperature impairs grain filling by inhibiting the deposition of storage materials such as starch and protein. To comprehend its impact on grain filling metabolism in rice (Oryza sativa), levels of metabolites and transcripts related to central pathways of metabolism were simultaneously determined in developing caryopses exposed to high temperature (33°C/28°C) and a control temperature (25°C/20°C) during the milky stage. A capillary electrophoresis-based metabolomic analysis revealed that high temperature increased the accumulation of sucrose and pyruvate/ oxaloacetate-derived amino acids and decreased levels of sugar phosphates and organic acids involved in glycolysis/gluconeogenesis and the tricarboxylic acid (TCA) cycle, respectively. A transcriptomic analysis using a whole genome-covering microarray unraveled the possible metabolic steps causing the shortage of storage materials under the elevated temperature. Starch deposition might be impaired by down-regulation of sucrose import/degradation and starch biosynthesis, and/or up-regulation of starch degradation as well as inefficient ATP production by an inhibited cytochrome respiration chain, as indicated by the response of gene expression to high temperature. Amino acid accumulation might be attributed to the heat-stable import of amino acids into the caryopsis and/or repression of protein synthesis especially the tRNA charging step under high temperature. An atlas showing the effect of high temperature on levels of metabolites and gene expression in the central metabolic pathways is presented.

Exit Strategies for Charged tRNA from GluRS

For several class I aminoacyl-tRNA synthetases (aaRSs), the rate-determining step in aminoacylation is the dissociation of charged tRNA from the enzyme. In this study, the following factors affecting the release of the charged tRNA from aaRSs are computationally explored: the protonation states of amino acids and substrates present in the active site, and the presence and the absence of AMP and elongation factor Tu.Through molecular modeling, internal pKa calculations, and molecular dynamics simulations, distinct, mechanistically relevant post-transfer states with charged tRNA bound to glutamyl-tRNA synthetase from Thermus thermophilus (Glu-tRNAGlu) are considered. The behavior of these nonequilibrium states is characterized as a function of time using dynamical network analysis, local energetics, and changes in free energies to estimate transitions that occur during the release of the tRNA. The hundreds of nanoseconds of simulation time reveal system characteristics that are consistent with recent experimental studies.Energetic and network results support the previously proposed mechanism in which the transfer of amino acid to tRNA is accompanied by the protonation of AMP to H-AMP. Subsequent migration of proton to water reduces the stability of the complex and loosens the interface both in the presence and in the absence of AMP. The subsequent undocking of AMP or tRNA then proceeds along thermodynamically competitive pathways. Release of the tRNA acceptor stem is further accelerated by the deprotonation of the α-ammonium group on the charging amino acid. The proposed general base is Glu41, a residue binding the α-ammonium group that is conserved in both structure and sequence across nearly all class I aaRSs. This universal handle is predicted through pKa calculations to be part of a proton relay system for destabilizing the bound charging amino acid following aminoacylation. Addition of elongation factor Tu to the aaRS·tRNA complex stimulates the dissociation of the tRNA core and the tRNA acceptor stem.

Ligand-bound Structures Provide Atomic Snapshots for the Catalytic Mechanism of d-Amino Acid Deacylase

d-tyrosyl-tRNATyr deacylase (DTD) is an editing enzyme that removes d-amino acids from mischarged tRNAs. We describe an in-depth analysis of the malaria parasite Plasmodium falciparum DTD here. Our data provide structural insights into DTD complexes with adenosine and d-amino acids. Bound adenosine is proximal to the DTD catalysis site, and it represents the authentic terminal adenosine of charged tRNA. DTD-bound d-amino acids cluster at three different subsites within the overall active site pocket. These subsites, called transition, active, and exit subsites allow docking, re-orientation, chiral selection, catalysis, and exit of the free d-amino acid from DTD. Our studies reveal variable modes of d-amino acid recognition by DTDs, suggesting an inherent plasticity that can accommodate all d- amino acids. An in-depth analysis of native, ADP-bound, and d- amino acid-complexed DTD structures provide the first atomic snapshots of ligand recognition and subsequent catalysis by this enzyme family. We have mapped sites for the deacylation reaction and mark possible routes for entry and egress of all substrates and products. We have also performed structure-based inhibitor discovery and tested lead compounds against the malaria parasite P. falciparum using growth inhibition assays. Our studies provide a comprehensive structural basis for the catalytic mechanism of DTD enzymes and have implications for inhibition of this enzyme in P. falciparum as a route to inhibiting the parasite.

Top-Down Tandem Mass Spectrometry of tRNA Via Ion Trap Collision-Induced Dissociation

Transfer RNA is a class of highly modified and structured non-coding RNA molecules generally comprised of 74-95 nucleotides. In this study, tandem mass spectrometry of intact multiply charged tRNA anions of roughly 25 kDa in mass has been demonstrated using a quadrupole/time-of-flight tandem mass spectrometer adapted for ion/ion reaction studies. The sample proved to be a mixture of tRNA molecules. The mass of the most abundant component of the mixture was not consistent with that of the nominal identity of the tRNA from the supplier, viz., tRNA(Phe); rather, the mass was consistent with tRNA(Phe) bearing an incomplete 3'-terminus. Multiply-charged anions from the major components were isolated in the gas phase and subjected to ion trap collision-induced dissociation without subsequent ion/ion reactions. Abundant fragments from the 5'- and 3'-termini of the molecule could be used to identify the major component as tRNA(Phe)-3'adenosine (without 3'-phosphorylation). Roughly 15% of the primary sequence of the intact tRNA was unambiguously reflected in the product ion spectrum. The existence of a possible tRNA(Phe) variant and the intact tRNA(Phe) was also supported by ion trap CID data. The multiply-charged fragment ions derived from tRNA(Phe)-3'adenosine were further charge-reduced to mostly singly- and doubly-charged species via proton transfer ion/ion reactions with benzoquinoline cations. The resulting reduction in spectral overlap and charge state ambiguity simplified interpretation of the product ion spectrum and allowed for the identification of product ions from roughly 60% of the sequence.

A glycine hinge for tRNA-dependent translocation of editing substrates to prevent errors by leucyl-tRNA synthetase

Aminoacyl-tRNA synthetases often rely on a proofreading mechanism to clear mischarging errors before they can be incorporated into newly synthesized proteins. Leucyl-tRNA synthetase (LeuRS) houses a hydrolytic editing pocket in a domain that is distinct from its aminoacylation domain. Mischarged amino acids are transiently translocated ∼30 Å between active sites for editing by an unknown tRNA-dependent mechanism. A glycine within a flexible β-strand that links the aminoacylation and editing domains of LeuRS was determined to be important to tRNA translocation. The translocation-defective mutation also demonstrated that the editing site screens both correctly and incorrectly charged tRNAs prior to product release.

Genome-wide Analysis of tRNA Charging and Activation of the eIF2 Kinase Gcn2p

When cells are subjected to nutritional stress, uncharged tRNAs accumulate and activate Gcn2p phosphorylation of eukaryotic initiation factor-2 (eIF2) and the general amino acid control pathway. The Gcn2p regulatory domain homologous to histidyl-tRNA synthetases is proposed to bind to uncharged tRNA, directly contributing to activation of Gcn2p. Here we apply a microarray technology to analyze genome-wide changes in tRNA charging in yeast upon activation of Gcn2p in response to amino acid starvation and high salinity, a stress not directly linked to nutritional deficiency. This microarray technology is applicable for all eukaryotic cells. Strains were starved for histidine, leucine, or tryptophan and shown to rapidly induce Gcn2p phosphorylation of eIF2. The relative charging level of all tRNAs was measured before and after starvation, and Gcn2p activation and the intracellular levels of the starved amino acid correlate with the observed decrease in tRNA charging. Interestingly, in some cases, tRNAs not charged with the starved amino acid became deacylated more rapidly than tRNAs charged with the starved amino acid. This increase in uncharged tRNA levels occurred although the intracellular levels for these non-starved amino acids remained unchanged. Additionally, treatment of a wild-type strain with high salinity stress showed transient changes in the charging of several different tRNAs. These results suggest that Gcn2p can be activated by many different tRNA species in the cell. These results also depict a complex cellular relationship between tRNA charging, amino acid availability, and non-nutrient stress. These relationships are best revealed by simultaneous monitoring of the charging level of all tRNAs.

Eukaryotic cytosolic and mitochondrial phenylalanyl-tRNA synthetases catalyze the charging of tRNA with the meta -tyrosine

The accumulation of proteins damaged by reactive oxygen species (ROS), conventionally regarded as having pathological potentials, is associated with age-related diseases such as Alzheimer's, atherosclerosis, and cataractogenesis. Exposure of the aromatic amino acid phenylalanine to ROS-generating systems produces multiple isomers of tyrosine: m-tyrosine (m-Tyr), o-tyrosine (o-Tyr), and the standard p-tyrosine (Tyr). Previously it was demonstrated that exogenously supplied, oxidized amino acids could be incorporated into bacterial and eukaryotic proteins. It is, therefore, likely that in many cases, in vivo-damaged amino acids are available for de novo synthesis of proteins. Although the involvement of aminoacyl-tRNA synthetases in this process has been hypothesized, the specific pathway by which ROS-damaged amino acids are incorporated into proteins remains unclear. We provide herein evidence that mitochondrial and cytoplasmic phenylalanyl-tRNA synthetases (HsmtPheRS and HsctPheRS, respectively) catalyze direct attachment of m-Tyr to tRNA(Phe), thereby opening the way for delivery of the misacylated tRNA to the ribosome and incorporation of ROS-damaged amino acid into eukaryotic proteins. Crystal complexes of mitochondrial and bacterial PheRSs with m-Tyr reveal the net of highly specific interactions within the synthetic and editing sites.

Charged tRNAs charge into secondary metabolism

Peptide bond formation is catalyzed from stable aminoacyl-tRNA intermediates by several specialized proteins. A new set of these enzymes forming cyclodipeptides present the first usage of tRNAs in secondary metabolism.