A protein extension to shorten RNA: elongated elongation factor-Tu recognizes the D-arm of T-armless tRNAs in nematode mitochondria

Nematode mitochondria possess extremely truncated tRNAs. Of 22 tRNAs, 20 lack the entire T-arm. The T-arm is necessary for the binding of canonical tRNAs and EF (elongation factor)-Tu (thermo-unstable). The nematode mitochondrial translation system employs two different EF-Tu factors named EF-Tu1 and EF-Tu2. Our previous study showed that nematode Caenorhabditis elegans EF-Tu1 binds specifically to T-armless tRNA. C. elegans EF-Tu1 has a 57-amino acid C-terminal extension that is absent from canonical EF-Tu, and the T-arm-binding residues of canonical EF-Tu are not conserved. In this study, the recognition mechanism of T-armless tRNA by EF-Tu1 was investigated. Both modification interference assays and primer extension analysis of cross-linked ternary complexes revealed that EF-Tu1 interacts not only with the tRNA acceptor stem but also with the D-arm. This is the first example of an EF-Tu recognizing the D-arm of a tRNA. The binding activity of EF-Tu1 was impaired by deletion of only 14 residues from the C-terminus, indicating that the C-terminus of EF-Tu1 is required for its binding to T-armless tRNA. These results suggest that C. elegans EF-Tu1 recognizes the D-arm instead of the T-arm by a mechanism involving its C-terminal region. This study sheds light on the co-evolution of RNA and RNA-binding proteins in nematode mitochondria.     

T-armless tRNAs and elongated elongation factor Tu

Most tRNAs share a common secondary structure containing a T arm, a D arm, an anticodon arm and an acceptor stem. However, there are some exceptions. Most nematode mitochondrial tRNAs and some animal mitochondrial tRNAs lack the T arm, which is necessary for binding to canonical elongation factor Tu (EF-Tu). The mitochondria of the nematode Caenorhabditis elegans have a unique EF-Tu, named EF-Tu1, whose structure has supplied clues as to how truncated tRNAs can work in translation. EF-Tu1 has a C-terminal extension of about 60 aa that is absent in canonical EF-Tu. Recent data from our laboratory strongly suggests that EF-Tu1 recognizes the D-arm instead of the T arm by a mechanism involving this C-terminal region. Further biochemical analysis of mitochondrial tRNAs and EF-Tu from the distantly related nematode Trichinella spp. and sequence information on nuclear and mitochondrial DNA in arthropods suggest that T-armless tRNAs may have arisen as a result of duplication of the EF-Tu gene. These studies provide valuable insights into the co-evolution of RNA and RNA-binding proteins.     

A unique serine-specific elongation factor Tu found in nematode mitochondria

The translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNAs to ribosomes by recognizing the tRNA acceptor and T stems. However, the unusual truncation observed in some animal mitochondrial tRNAs seems to prevent recognition by a canonical EF-Tu. For instance, nematode mitochondria contain tRNAs lacking a T or D arm. We recently found an atypical EF-Tu (EF-Tu1) specific for nematode mitochondrial tRNAs that lack the T arm. We have now discovered a second factor, EF-Tu2, which binds only to tRNAs that lack a D arm. EF-Tu2 seems unique in its amino acid specificity because it recognizes the aminoacyl moiety of seryl-tRNAs and the tRNA structure itself. Such EF-Tu evolution might explain tRNA structural divergence in animal mitochondria.     

Characterization of the interaction between the nucleotide exchange factor EF-Ts from nematode mitochondria and elongation factor Tu

Caenorhabditis elegans mitochondria have two elongation factor (EF)-Tu species, denoted EF-Tu1 and EF-Tu2. Recombinant nematode EF-Ts purified from Escherichia coli bound both of these molecules and also stimulated the translational activity of EF-Tu, indicating that the nematode EF-Ts homolog is a functional EF-Ts protein of mitochondria. Complexes formed by the interaction of nematode EF-Ts with EF-Tu1 and EF-Tu2 could be detected by native gel electrophoresis and purified by gel filtration. Although the nematode mitochondrial (mt) EF-Tu molecules are extremely unstable and easily form aggregates, native gel electrophoresis and gel filtration analysis revealed that EF-Tu·EF-Ts complexes are significantly more soluble. This indicates that nematode EF-Ts can be used to stabilize homologous EF-Tu molecules for experimental purposes. The EF-Ts bound to two eubacterial EF-Tu species (E.coli and Thermus thermophilus). Although the EF-Ts did not bind to bovine mt EF-Tu, it could bind to a chimeric nematode–bovine EF-Tu molecule containing domains 1 and 2 from bovine mt EF-Tu. Thus, the nematode EF-Ts appears to have a broad specificity for EF-Tu molecules from different species.     

Intricacies and surprises of nuclear-mitochondrial co-evolution

In this issue of the Biochemical Journal, Watanabe and colleagues disclose another fascinating facet of the mitochondrial protein synthesis machinery: one of the two nematode mitochondrial elongation factors Tu, EF-Tu1, specifically recognizes the D-arm of T-armless tRNAs via a 57-amino-acid C-terminal extension that compensates for the reduction in tRNA structure. This principle provides a paradigm for the evolutionary events thought to have ignited the transition from an ancient 'RNA world' to the 'protein world' of today.     

Kinetic Analysis Suggests Evolution of Ribosome Specificity in Modern Elongation Factor-Tus from “Generalist” Ancestors

It has been hypothesized that early enzymes are more promiscuous than their extant orthologs. Whether or not this hypothesis applies to the translation machinery, the oldest molecular machine of life, is not known. Efficient protein synthesis relies on a cascade of specific interactions between the ribosome and the translation factors. Here, using elongation factor-Tu (EF-Tu) as a model system, we have explored the evolution of ribosome specificity in translation factors. Employing presteady state fast kinetics using quench flow, we have quantitatively characterized the specificity of two sequence-reconstructed 1.3- to 3.3-Gy-old ancestral EF-Tus toward two unrelated bacterial ribosomes, mesophilic Escherichia coli and thermophilic Thermus thermophilus. Although the modern EF-Tus show clear preference for their respective ribosomes, the ancestral EF-Tus show similar specificity for diverse ribosomes. In addition, despite increase in the catalytic activity with temperature, the ribosome specificity of the thermophilic EF-Tus remains virtually unchanged. Our kinetic analysis thus suggests that EF-Tu proteins likely evolved from the catalytically promiscuous, “generalist” ancestors. Furthermore, compatibility of diverse ribosomes with the modern and ancestral EF-Tus suggests that the ribosomal core probably evolved before the diversification of the EF-Tus. This study thus provides important insights regarding the evolution of modern translation machinery.     

A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2

Nematode mitochondria expresses two types of extremely truncated tRNAs that are specifically recognized by two distinct elongation factor Tu (EF-Tu) species named EF-Tu1 and EF-Tu2. This is unlike the canonical EF-Tu molecule that participates in the standard protein biosynthesis systems, which basically recognizes all elongator tRNAs. EF-Tu2 specifically recognizes Ser-tRNA^Ser that lacks a D arm but has a short T arm. Our previous study led us to speculate the lack of the D arm may be essential for the tRNA recognition of EF-Tu2. However, here, we showed that the EF-Tu2 can bind to D arm-bearing Ser-tRNAs, in which the D–T arm interaction was weakened by the mutations. The ethylnitrosourea-modification interference assay showed that EF-Tu2 is unique, in that it interacts with the phosphate groups on the T stem on the side that is opposite to where canonical EF-Tu binds. The hydrolysis protection assay using several EF-Tu2 mutants then strongly suggests that seven C-terminal amino acid residues of EF-Tu2 are essential for its aminoacyl-tRNA-binding activity. Our results indicate that the formation of the nematode mitochondrial (mt) EF-Tu2/GTP/aminoacyl-tRNA ternary complex is probably supported by a unique interaction between the C-terminal extension of EF-Tu2 and the tRNA.     

Identification of the residues involved in the unique serine specificity of Caenorhabditis elegans mitochondrial EF-Tu2

In canonical translation systems, the single elongation factor Tu (EF-Tu) recognizes all elongator tRNAs. However, in Caenorhabditis elegans mitochondria, two distinct EF-Tu species, EF-Tu1 and EF-Tu2, recognize 20 species of T armless tRNA and two species of D armless tRNA(Ser), respectively. We previously reported that C. elegans mitochondrial EF-Tu2 specifically recognizes the serine moiety of serylated-tRNA. In this study, to identify the critical residues for the serine specificity in EF-Tu2, several residues in the amino acid binding pocket of bacterial EF-Tu were systematically replaced with corresponding EF-Tu2 residues, and the mutants were analyzed for their specificity for esterified amino acids attached to tRNAs. In this way, we obtained a bacterial EF-Tu mutant that acquired serine specificity after the introduction of 10 EF-Tu2 residues into its amino acid binding pocket. C. elegans EF-Tu2 mutants lacking serine specificity were also created by replacing seven or eight residues with bacterial residues. Further stressing the importance of these residues, we found that they are almost conserved in EF-Tu2 sequences of closely related nematodes. Thus, these three approaches reveal the critical residues essential for the unique serine specificity of C. elegans mitochondrial EF-Tu2.     

An "elongated" translation elongation factor Tu for truncated tRNAs in nematode mitochondria

We have found the gene for a translation elongation factor Tu (EF-Tu) homologue in the genome of the nematode Caenorhabditis elegans. Because the corresponding protein was detected immunologically in a nematode mitochondrial (mt) extract, it could be regarded as a nematode mt EF-Tu. The protein possesses an extension of about 57 amino acids (we call this domain 3') at the C terminus, which is not found in any other known EF-Tu. Because most nematode mt tRNAs lack a T stem, domain 3' may be related to this feature. The nematode EF-Tu bound to nematode T stem-lacking tRNA, but bacterial EF-Tu was unable to do so. A series of domain exchange experiments strongly suggested that domains 3 and 3' are essential for binding to T stem-lacking tRNAs. This finding may constitute a novel example of the co-evolution of a structurally simplified RNA and the cognate RNA-binding protein, the latter having apparently acquired an additional domain to compensate for the lack of a binding site(s) on the RNA.     

Structural elements defining elongation factor Tu mediated suppression of codon ambiguity

In most prokaryotes Asn-tRNA^Asn and Gln-tRNA^Gln are formed by amidation of aspartate and glutamate mischarged onto tRNA^Asn and tRNA^Gln, respectively. Coexistence in the organism of mischarged Asp-tRNA^Asn and Glu-tRNA^Gln and the homologous Asn-tRNA^Asn and Gln-tRNA^Gln does not, however, lead to erroneous incorporation of Asp and Glu into proteins, since EF-Tu discriminates the misacylated tRNAs from the correctly charged ones. This property contrasts with the canonical function of EF-Tu, which is to non-specifically bind the homologous aa-tRNAs, as well as heterologous species formed in vitro by aminoacylation of non-cognate tRNAs. In Thermus thermophilus that forms the Asp-tRNA^Asn intermediate by the indirect pathway of tRNA asparaginylation, EF-Tu must discriminate the mischarged aminoacyl-tRNAs (aa-tRNA). We show that two base pairs in the tRNA T-arm and a single residue in the amino acid binding pocket of EF-Tu promote discrimination of Asp-tRNA^Asn from Asn-tRNA^Asn and Asp-tRNA^Asp by the protein. Our analysis suggests that these structural elements might also contribute to rejection of other mischarged aa-tRNAs formed in vivo that are not involved in peptide elongation. Additionally, these structural features might be involved in maintaining a delicate balance of weak and strong binding affinities between EF-Tu and the amino acid and tRNA moieties of other elongator aa-tRNAs.     

Characterization of cholinesterases from the parasitic nematode Trichinella spiralis

1. Trichinella cholinesterases occur in multiple molecular forms which differ in size, kinetics, activity with butyrylthiocholine, and effects of inhibitors.2. The 5.3 and 13S forms identified in Trichinella extracts are also found in C. elegans and other nematodes but the 7S form which occurs in other nematodes was absent from Trichinella detergent extracts. Differences in kinetic and inhibition properties among nematode species were also evident.3. The level of cholinesterases in excretory/secretory products is low.4. Trichinella cholinesterases did not elicit a detectable antibody response in mice.     

Steady-state levels of imported tRNAs in Chlamydomonas mitochondria are correlated with both cytosolic and mitochondrial codon usages

The mitochondrial genome of Chlamydomonas reinhardtii only encodes three expressed tRNA genes, thus most mitochondrial tRNAs are likely imported. The sharing of tRNAs between chloroplasts and mitochondria has been speculated in this organism. We first demonstrate that no plastidial tRNA is present in mitochondria and that the mitochondrial translation mainly relies on the import of nucleus-encoded tRNA species. Then, using northern analysis, we show that the extent of mitochondrial localization for the 49 tRNA isoacceptor families encoded by the C. reinhardtii nuclear genome is highly variable. Until now the reasons for such variability were unknown. By comparing cytosolic and mitochondrial codon usage with the sub-cellular distribution of tRNAs, we provide unprecedented evidence that the steady-state level of a mitochondrial tRNA is linked not only to the frequency of the cognate codon in mitochondria but also to its frequency in the cytosol, then allowing optimal mitochondrial translation.     

Mitochondrial translation: elongation factor tu is essential in fission yeast and depends on an exchange factor conserved in humans but not in budding yeast

The translation elongation factor EF-Tu is a GTPase that delivers amino-acylated tRNAs to the ribosome during the elongation step of translation. EF-Tu/GDP is recycled by the guanine nucleotide exchange factor EF-Ts. Whereas EF-Ts is lacking in S. cerevisiae, both translation factors are found in S. pombe and H. sapiens mitochondria, consistent with the known similarity between fission yeast and human cell mitochondrial physiology. We constructed yeast mutants lacking these elongation factors. We show that mitochondrial translation is vital for S. pombe, as it is for human cells. In a genetic background allowing the loss of mitochondrial functions, a block in mitochondrial translation in S. pombe leads to a major depletion of mtDNA. The relationships between EF-Ts and EF-Tu from both yeasts and humans were investigated through functional complementation and coexpression experiments and by a search for suppressors of the absence of the S. pombe EF-Ts. We find that S. cerevisiae EF-Tu is functionally equivalent to the S. pombe EF-Tu/EF-Ts couple. Point mutations in the S. pombe EF-Tu can render it independent of its exchange factor, thereby mimicking the situation in S. cerevisiae.     

Uniform affinity-tuning of N-methyl-aminoacyl-tRNAs to EF-Tu enhances their multiple incorporation

In ribosomal translation, the accommodation of aminoacyl-tRNAs into the ribosome is mediated by elongation factor thermo unstable (EF-Tu). The structures of proteinogenic aminoacyl-tRNAs (pAA-tRNAs) are fine-tuned to have uniform binding affinities to EF-Tu in order that all proteinogenic amino acids can be incorporated into the nascent peptide chain with similar efficiencies. Although genetic code reprogramming has enabled the incorporation of non-proteinogenic amino acids (npAAs) into the nascent peptide chain, the incorporation of some npAAs, such as N-methyl-amino acids (^MeAAs), is less efficient, especially when ^MeAAs frequently and/or consecutively appear in a peptide sequence. Such poor incorporation efficiencies can be attributed to inadequate affinities of ^MeAA-tRNAs to EF-Tu. Taking advantage of flexizymes, here we have experimentally verified that the affinities of ^MeAA-tRNAs to EF-Tu are indeed weaker than those of pAA-tRNAs. Since the T-stem of tRNA plays a major role in interacting with EF-Tu, we have engineered the T-stem sequence to tune the affinity of ^MeAA-tRNAs to EF-Tu. The uniform affinity-tuning of the individual pairs has successfully enhanced the incorporation of ^MeAAs, achieving the incorporation of nine distinct ^MeAAs into both linear and thioether-macrocyclic peptide scaffolds.     

Pathology-related substitutions in human mitochondrial tRNA(Ile) reduce precursor 3' end processing efficiency in vitro

The human mitochondrial genome encodes 22 tRNAs interspersed among the two rRNAs and 11 mRNAs, often without spacers, suggesting that tRNAs must be efficiently excised. Numerous maternally transmitted diseases and syndromes arise from mutations in mitochondrial tRNAs, likely due to defect(s) in tRNA metabolism. We have systematically explored the effect of pathogenic mutations on tRNA^Ile precursor 3′ end maturation in vitro by 3′-tRNase. Strikingly, four pathogenic tRNA^Ile mutations reduce 3′-tRNase processing efficiency (V_max / K_M) to ~10-fold below that of wild-type, principally due to lower V_max. The structural impact of mutations was sought by secondary structure probing and wild-type tRNA^Ile precursor was found to fold into a canonical cloverleaf. Among the mutant tRNA^Ile precursors with the greatest 3′ end processing deficiencies, only G4309A displays a secondary structure substantially different from wild-type, with changes in the T domain proximal to the substitution. Reduced efficiency of tRNA^Ile precursor 3′ end processing, in one case associated with structural perturbations, could thus contribute to human mitochondrial diseases caused by mutant tRNAs.     

The Complete Mitochondrial Genome of the Entomopathogenic Nematode Steinernema carpocapsae: Insights into Nematode Mitochondrial DNA Evolution and Phylogeny

We determined the complete sequence of the mitochondrial DNA of the entomopathogenic nematode Steinernema carpocapsae and analyzed its structure and composition as well as the secondary structures predicted for its tRNAs and rRNAs. Almost the complete genome has been amplified in one fragment with long PCR and sequenced using a shotgun strategy. The 13,925-bp genome contains genes for 2 rRNAs, 22 tRNAs, and 12 proteins and lacks an ORF encoding ATPase subunit 8. Four initiation codons were inferred, TTT, TTA, ATA, and ATT, most of the genes ended with TAA or TAG, and only two had a T as an incomplete stop codon. All predicted tRNAs showed the nonconventional secondary structure typical of Secernentea. Although we were able to fold the sequences of trnN, trnD, and trnC into more conventional cloverleaf structures after adding adjacent nucleotides, northern blot experiments showed that the nonstandard tRNAs are actually expressed. Phylogenetic and comparative analyses showed that the mitochondrial genome of S. carpocapsae is more closely related to the genomes of A. suum and C. elegans than to that of Strongyloides stercoralis. This finding does not support the phylogeny based on nuclear small subunit ribosomal DNA sequences previously published. This discrepancy may result from differential reproductive strategies and/or differential selective pressure acting on nuclear and mitochondrial genes. The distinctive characteristics observed among mitochondrial genomes of Secernentea may have arisen to counteract the deleterious effects of Muller’s ratchet, which is probably enhanced by the reproductive strategies and selective pressures referred to above. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

Caenorhabditis elegans neuron degeneration and mitochondrial suppression caused by selected environmental chemicals

Mitochondrial alterations have been documented for many years in the brains of Parkinson’s disease (PD), a disorder that is characterized by the selective loss of dopamine neurons. Recent studies have demonstrated that Parkinson’s disease-associated proteins are either present in mitochondria or translocated into mitochondria in response to stress, further reinforcing the importance of the mitochondrial function in the pathogenesis of Parkinson’s disease. Exposure to environmental chemicals such as pesticides and heavy metals has been suggested as risk factors in the development of Parkinson’s disease. It has been reported that a number of environmental agents including tobacco smoke and perfluorinated compounds, pesticides, as well as metals (Mn²⁺ and Pb²⁺) modulate mitochondrial function. However the exact mechanism of mitochondrial alteration has not been defined in the context of the development and progression of Parkinson’s disease. The complexity of the mammalian system has made it difficult to dissect the molecular components involved in the pathogenesis of Parkinson’s disease. In the present study we used the nematode Caenorhabditis elegans (C. elegans) model of neuron degeneration and investigated the effect of environmental chemicals on mitochondrial biogenesis and mitochondrial gene regulation. Chronic exposure to low concentration (2 or 4 μM) of pesticide rotenone, resulted in significant loss of dopamine neuron in C. elegans, a classic feature of Parkinson’s disease. We then determined if the rotenone-induced neuron degeneration is accompanied by a change in mitochondria biogenesis. Analysis of mitochondrial genomic replication by quantitative PCR showed a dramatic decrease in mitochondrial DNA (mtDNA) copies of rotenone-treated C. elegans compared to control. This decreased mitochondrial biogenesis occurred prior to the development of loss of dopamine neurons, and was persistent. The inhibition of mtDNA replication was also found in C. elegans exposed to another neuron toxicant Mn²⁺ at the concentration 50 or 100 mM. We further examined the mitochondrial gene expression and found significant lower level of mitochondrial complex IV subunits COI and COII in C. elegans exposed to rotenone. These results demonstrate that environmental chemicals cause persistent suppression of mitochondrial biogenesis and mitochondrial gene expression, and suggest a critical role of modifying mitochondrial biogenesis in toxicants-induced neuron degeneration in C. elegans model.     

Mixed infection, Trichinella spiralis and Trichinella britovi, in a wild boar hunted in the Province of Cáceres (Spain)

The etiological agents of human trichinellosis are distributed worldwide in domestic and wild animals. In Spain, two morphologically indistinguishable Trichinella species have been described—Trichinella spiralis and Trichinella britovi—that are perpetuated in both domestic and sylvatic cycles. The present work reports a double natural infection involving these species in a wild boar killed by hunters in the Province of Cáceres, Spain. After artificial digestion of the boar’s muscles, nine larvae/g were collected. These were characterized by multiplex-PCR and Western-blotting using the Trichinella-specific monoclonal antibodies US5 and US9, and both T. spiralis and T. britovi were detected. The mechanism by which this wild boar came to acquire a mixed infection remains unclear.