Characterization of regular polymerization products of elongation factor EF-Tu from Escherichia coli by electron microscopy and image processing

Regular cylindrical polymers of the elongation factor EF-Tu from Escherichia coli have been prepared and their structural parameters have been determined by electron microscopy and image processing. The analysis yielded information regarding the packaging of the EF-Tu · GDP monomers within the polymers and their low resolution structure. Interestingly, the structural integrity of the EF-Tu molecule determines the type of polymerization products formed. Intact EF-Tu · GDP polymerizes into cylindrical structures with a diameter of 150 Å and a repeat distance of about 265 Å. The cylindrical symmetry was found to be 5-fold. After specific cleavage of the protein chain close to the N-terminus, EF-Tu · GDP associates to cylindrical polymers of a much larger radius (diameter about 300 Å). In this case the indexing of the optical transforms could not be carried out unambiguously. In addition to the linear polymers, non-linear assemblies of intact EF-Tu · GDP have also been detected. All of the resulting polymerization products retained most of the biological activity. This polymerization was inhibited by the presence of the antibiotic aurodox, which suggests that only the EF-Tu in the GDP-like conformation is able to polymerize under these circumstances.The present study illustrates that the multi-functional protein EF-Tu, which can undergo various allosteric transitions, can assemble into different supramolecular structures.     

Structural model for the selenocysteine-specific elongation factor SelB

A structural model was established for the N-terminal part of translation factor SelB which shares sequence similarity with EF-Tu, taking into account the coordinates of the EF-Tu 3D structure and the consensus of SelB sequences from four bacteria. The model showed that SelB is homologous in its N-terminal domains over all three domains of EF-Tu. The guanine nucleotide binding site and the residues involved in GTP hydrolysis are similar to those of EF-Tu, but with some subtle differences possibly responsible for the higher affinity of SelB for GTP compared to GDP. In accordance, the EF-Tu epitopes interacting with EF-Ts are lacking in SelB. Information on the formation of the selenocysteyl-binding pocket is presented. A phylogenetic comparison of the SelB domains homologous to EF-Tu with those from EF-Tu and initiation factor 2 indicated that SelB forms a separate class of translation factors.     

Cytoskeletal elements in bacteria Mycoplasma pneumoniae, Thermoanaerobacterium sp., and Escherichia coli as revealed by electron microscopy

Recently, electron microscopic studies on the eubacteria Mycoplasma pneumoniae, Thermoanaerobacterium sp., and Escherichia coli have revealed the existence of cytoskeletal elements so far unknown in prokaryotes. The wall-less bacterium M. pneumoniae contains, in close vicinity to the inner face of the cytoplasmic membrane, a helically organized lining composed of protein elements that form a regular network of meshes that encloses the entire cytoplasm. Numerous regularly spaced pin-like structural elements, the stalks with terminal knobs, connect the lining with the cytoplasmic membrane. In this bacterium, a specific rod-like structural element is located in the tip region. Occasionally, it is bent or twisted. It consists of two matching blade-like sub-elements. A number of parallel linkers, extending from the edges of the rod, make contact with the lining. The proximal end of the rod is attached to a wheel-like complex. Fibrils originating from the wheel cross the cytoplasm and make contact with the lining. E. coli contains a similar helically organized lining close to the inner face of the cytoplasmic membrane. Groups of ribosomes (polysomes) were seen to be attached to the helical elements of the lining. A feature that is common to both bacteria and to Thermoanaerobacterium sp. appears to be that the lining and the fibrils crossing the cytoplasm contain a high number of copies of the bacterial elongation factor Tu (EF-Tu). This indicates that this protein may play an important role as a structural element in bacterial cytoskeletons. This notion was supported by experiments in which the cytoskeleton in E. coli was destabilized by induced expression of truncated EF-Tu, with the consequence of cell lysis, and by the finding that in vitro polymerization of monomeric EF-Tu into protofilaments was hindered in a mixture of full-size EF-Tu and truncated EF-Tu consisting of domain 3 only. Current research and developmental efforts are aimed at the design of a new class of antibacterial drugs, acting by destabilization of the EF-Tu-containing bacterial cytoskeleton, and of an innovative mode of inducible lysis of recombinant bacteria by controlled destabilization of the EF-Tu-containing cytoskeleton.     

Isolation and characterization of Streptomyces aureofaciens protein-synthesis elongation factor Tu in an aggregated state

The ability of EF-Tu to aggregate spontaneously was employed for the purification of homogeneous EF-Tu . GDP from Streptomyces aureofaciens. The formation of filamentous structures in the aggregated EF-Tu was demonstrated in a light microscope. The purified factor, with a specific activity of 19,100 +/- 1,000 units/mg in [3H]GDP exchange, was shown to be active in the translation of poly(U). Aggregated EF-Tu . GDP exhibited almost eight-times lower GDP-exchange capacity at 2 degrees C than at 30 degrees C. This suggests that GDP-binding sites are not freely accessible at lower temperatures in the aggregated factor, in contrast to Escherichia coli polymerized EF-Tu. Turbidimetric assays revealed that the solubilization of diluted aggregated S. aureofaciens EF-Tu is strongly dependent on temperature and causes an increase in the number of accessible GDP-binding sites.     

Effects of mutagenesis of residue 221 on the properties of bacterial and mitochondrial elongation factor EF-Tu

During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of ribosomes. This factor is highly conserved throughout evolution. However, several key residues differ between bacterial and mammalian mitochondrial EF-Tu (EF-Tu(mt)). One such residue is Ser221 (Escherichia coli numbering). This residue is conserved as a Ser or Thr in the bacterial factors but is present as Pro269 in EF-Tu(mt). Pro269 reorients the loop containing this residue and shifts the adjoining beta-strand in EF-Tu(mt) compared to that of E. coli EF-Tu potentially altering the binding pocket for the acceptor stem of the aa-tRNA. Pro269 was mutated to a serine residue (P269S) in EF-Tu(mt). For comparison, the complementary mutation was created at Ser221 in E. coli EF-Tu (S221P). The E. coli EF-Tu S221P variant is poorly expressed in E. coli and the majority of the molecules fail to fold into an active conformation. In contrast, EF-Tu(mt) P269S is expressed to a high level in E. coli. When corrected for the percentage of active molecules, both variants function as effectively as their respective wild-type factors in ternary complex formation using E. coli Phe-tRNA(Phe) and Cys-tRNA(Cys). They are also active in A-site binding and in vitro translation assays with E. coli Phe-tRNA(Phe). In addition, both variants are as active as their respective wild-type factors in ternary complex formation, A-site binding and in vitro translation assays using mitochondrial Phe-tRNA(Phe).     

Structural homology between elongation factors EF-Tu from Bacillus stearothermophilus and Escherichia coli in the binding site for aminoacyl-tRNA

Elongation factor EF-Tu (Mr approximately equal to 50 000) and elongation factor EF-G (Mr approximately equal to 78 000) were isolated from Bacillus stearothermophilus in a homogeneous form. The ability of EF-Tu to participate in protein synthesis is rapidly inactivated by N-tosyl-L-phenyl-alanylchloromethane (Tos-PheCH2Cl). EF-Tu X GTP is more susceptible to the inhibition by Tos-PheCH2Cl than is EF-Tu X GDP. Tos-PheCH2Cl forms a covalent equimolar complex with the factor by reacting with a cysteine residue in its molecule. The labelling of EF-Tu by the reagent irreversibly destroys its ability to bind aminoacyl-tRNA, which in turn protects the protein from this inactivation. This indicates that the modification of EF-Tu by Tos-PheCH2Cl occurs at the aminoacyl-tRNA binding site of the protein. To identify and characterize the site of aminoacyl-tRNA binding in EF-Tu, the factor was labelled with [14C]Tos-PheCH2Cl, digested with trypsin, the resulting peptides were separated by high-performance liquid chromatography and the sequence of the radioactive peptide was determined. The peptide has identical structure with an Escherichia coli EF-Tu tryptic peptide comprising the residues 75-89 and the Tos-PheCH2Cl-reactive cysteine at position 81 [Jonák, J., Petersen, T. E., Clark, B. F. C. and Rychlík, I. (1982) FEBS Lett. 150, 485-488]. Experiments on photo-oxidation of EF-Tu by visible light in the presence of rose bengal dye showed that there are apparently two histidine residues in elongation factor Tu from B. stearothermophilus which are essential for the interaction with aminoacyl-tRNA. This is clearly reminiscent of a similar situation in E. coli EF-Tu [Jonák, J., Petersen, T. E., Meloun, B. and Rychlík, I. (1984) Eur. J. Biochem. 144, 295-303]. Our results provide further evidence for the conserved nature of the site of aminoacyl-tRNA binding in elongation factor EF-Tu and show that Tos-PheCH2Cl reagent might be a favourable tool for the identification of the site in the structure of prokaryotic EF-Tus.     

Methylation in vivo of elongation factor EF-Tu at lysine-56 decreases the rate of tRNA-dependent GTP hydrolysis

In this paper we show, that the in vivo methylation of the elongation factor Tu from Escherichia coli is correlated with the growth phase of the bacterium. Methylation occurs at one position only, i.e. Lys-56, and initially results in monomethylation during logarithmic growth. Upon entering the stationary phase of E. coli, monomethyllysine is gradually converted into dimethyllysine. We have undertaken an extensive comparison between the properties of the highly methylated EF-Tu and unmodified EF-Tu. No gross conformational differences, as measured by the rate of mild tryptic cleavage, were observed. The dissociation rates of the nucleotides GDP and GTP appear likewise to be unaffected by the methylation, just as is the stimulatory effect of the elongation factor Ts upon these rates. Whereas tRNA binding at the classical binding site of EF-Tu (site I) also appears not to be affected by the methylation of the protein, tRNA binding at site II is. Although the apparent affinity of tRNA for site II remains unaltered upon methylation of EF-Tu, the conformational effects of tRNA binding at this site become different. Both the GTPase activity of the protein and the reactivity of Cys-81 are significantly less stimulated by the tRNA when EF-Tu is methylated. A possible physiological implication of this phenomenon is discussed.     

Copolymerization of pNcollagen III and collagen I. pNcollagen III decreases the rate of incorporation of collagen I into fibrils, the amount of collagen I incorporated, and the diameter of the fibrils formed

Previous observations suggested that pNcollagen III, the partially processed form of type III procollagen, coats fibrils of collagen I and thereby helps regulate the diameter of fibrils formed by collagen I. The previous observations, however, did not exclude the possibility that pNcollagen III was deposited on preformed collagen I fibrils after the fibrils were assembled. Here, mixtures of pNcollagen III and collagen I were generated simultaneously by enzymatic cleavage of precursor forms of the proteins. The results demonstrated that pNcollagen III forms true copolymers with collagen I. The presence of pNcollagen III both inhibited the rate at which collagen I assembled into fibrils and decreased the amount of collagen I incorporated into fibrils at steady-state equilibrium. In addition, the results demonstrated that copolymerization of pNcollagen III with collagen I generated fibrils that were thinner than fibrils generated under the same conditions from collagen I alone. Increasing the initial molar ratio of pNcollagen III to collagen I in the solution-phase increased the amount of pNcollagen III copolymerizing with collagen I and progressively decreased the diameter of the fibrils. Therefore, the copolymers were heterogeneous in that the stoichiometry of the two monomers in the fibrils varied. The results are consistent with a model in which pNcollagen III can regulate the diameter of collagen I fibrils by coating the surface of the fibrils and thereby allow tip growth but not lateral growth of the fibrils.     

Elongation factor Tu of the extreme thermophilic hydrogen oxidizing bacterium Calderobacterium hydrogenophilum

Protein synthesis elongation factor Tu has been purified from an extreme thermophilic hydrogen oxidizing bacterium Calderobacterium hydrogenophilum. The molecular mass of EF-Tu. GDP is 51,000. The factor is heat stable and loses only 50% of its activity after heating for 5 min at 80 degrees C. Under mild conditions trypsin cleaved EF-Tu. GDP to four main fragments. Only one fragment of Mr = 20,000 had a mobility similar to the trypsin fragment "B" of Escherichia coli EF-Tu. Other peptide fragments of E. coli and C. hydrogenophilum EF-Tu differed in size, but native preparations of both factors are immunologically similar.     

Possible evolution of factors involved in protein biosynthesis.

The elongation factors of protein biosynthesis are well preserved through out evolution. They catalyze the elongation phase of protein biosynthesis, where on the ribosome amino acids are added one at a time to a growing peptide according to the genetic information transcribed into mRNA. Elongation factor Tu (EF-Tu) provides the binding of aminoacylated tRNA to the ribosome and protects the aminoester bond against hydrolysis until a correct match between the codon on mRNA and the anticodon on tRNA can be achieved. Elongation factor G (EF-G) supports the translocation of tRNAs and of mRNA on the ribosome so that a new codon can be exposed for decoding. Both these factors are GTP binding proteins, and as such exist in an active form with GTP and an inactive form with GDP bound to the nucleotide binding domain. Elongation factor Ts (EF-Ts) will catalyze the exchange of nucleotide on EF-Tu. This review describes structural work on EF-Tu performed in our laboratory over the last eight years. The structural results provide a rather complete picture of the major structural forms of EF-Tu, including the so called ternary complex of aa-tRNA:EF-Tu:GTP. The structural comparison of this ternary complex with the structure of EF-G:GDP displays an unexpected macromolecular mimicry, where three domains of EF-G mimick the shape of the tRNA in the ternary complex. This observation has initiated much speculation on the evolution of all factors involved in protein synthesis, as well as on the details of the ribosomal function in one part of elongation.     

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.     

Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo.

The binding of the EF-Tu.GTP.aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23S ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23S rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as normal or mutant ribosomal protein S12 (rpsL). Together with wild-type EF-Tu, the 2661C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wild-type form). Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2661C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa). The lethal effect obtained by the combination of 2661C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain. In such a viable strain, 2661C had no effect on either the translation elongation rate or nonsense codon suppression. Our data suggest that the G base at position 2661 in 23S rRNA is important for binding of EF-Tu during protein synthesis in vivo. The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.     

Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu

Yeast mitochondrial elongation factor Tu (EF-Tu) was purified 200-fold from a mitochondrial extract of Saccharomyces cerevisiae to yield a single polypeptide of Mr = approximately 47,000. The factor was detected by complementation with Escherichia coli elongation factor G and ribosomes in an in vitro phenylalanine polymerization reaction. Mitochondrial EF-Tu, like E. coli EF-Tu, catalyzes the binding of aminoacyl-tRNA to ribosomes and possesses an intrinsic GTP hydrolyzing activity which can be activated either by kirromycin or by ribosomes. Kinetic and binding analyses of the interactions of mitochondrial EF-Tu with guanine nucleotides yielded affinity constants for GTP and GDP of approximately 5 and 25 microM, respectively. The corresponding affinity constants for the E. coli factor are approximately 0.3 and 0.003 microM, respectively. In keeping with these observations, we found that purified mitochondrial EF-Tu, unlike E. coli EF-Tu, does not contain endogenously bound nucleotide and is not stabilized by GDP. In addition, we have been unable to detect a functional counterpart to E. coli EF-Ts in extracts of yeast mitochondria and E. coli EF-Ts did not detectably stimulate amino acid polymerization with mitochondrial EF-Tu or enhance the binding of guanine nucleotides to the factor. We conclude that while yeast mitochondrial EF-Tu is functionally analogous to and interchangeable with E. coli EF-Tu, its affinity for guanine nucleotides and interaction with EF-Ts are quite different from those of E. coli EF-Tu.     

Regulation of translation of the head protein of T4 bacteriophage by specific binding of EF-Tu to a leader sequence

Recent evidence indicates that translation elongation factor Tu (EF-Tu) has a role in the cell in addition to its well established role in translation. The translation factor binds to a specific region called the Gol region close to the N terminus of the T4 bacteriophage major head protein as the head protein emerges from the ribosome. This binding was discovered because EF-Tu bound to Gol peptide is the specific substrate of the Lit protease that cleaves the EF-Tu between amino acid residues Gly59 and lle60, blocking phage development. These experiments raised the question of why the Gol region of the incipient head protein binds to EF-Tu, as binding to incipient proteins is not expected from the canonical role of EF-Tu. Here, we use gol-lacZ translational fusions to show that cleavage of EF-Tu in the complex with Gol peptide can block translation of a lacZ reporter gene fused translationally downstream of the Gol peptide that activated the cleavage. We propose a model to explain how binding of EF-Tu to the emerging Gol peptide could cause translation to pause temporarily and allow time for the leader polypeptide to bind to the GroEL chaperonin before translation continues, allowing cotranslation of the head protein with its insertion into the GroEL chaperonin chamber, and preventing premature synthesis and precipitation of the head protein. Cleavage of EF-Tu in the complex would block translation of the head protein and therefore development of the infecting phage. Experiments are presented that confirm two predictions of this model. Considering the evolutionary conservation of the components of this system, this novel regulatory mechanism could be used in other situations, both in bacteria and eukaryotes, where proteins are cotranslated with their insertion into cellular structures.     

Intrinsic fluorescence of elongation factor Tu in its complexes with GDP and elongation factor Ts

The intrinsic fluorescence properties of elongation factor Tu (EF-Tu) in its complexes with GDP and elongation factor Ts (EF-Ts) have been investigated. The emission spectra for both complexes are dominated by the tyrosine contribution upon excitation at 280 nm whereas excitation at 300 nm leads to exclusive emission from the single tryptophan residue (Trp-184) of EF-Tu. The fluorescence lifetime of this tryptophan residue in both complexes was investigated by using a multifrequency phase fluorometer which achieves a broad range of modulation frequencies utilizing the harmonic content of a mode-locked laser. These results indicated a heterogeneous emission with major components near 4.8 ns for both complexes. Quenching experiments on both complexes indicated limited accessibility of the tryptophan residue to acrylamide and virtually no accessibility to iodide ion. The quenching patterns exhibited by EF-Tu-GDP and EF-Tu X EF-Ts were, however, different; both quenchers were more efficient at quenching the emission from the EF-Tu x EF-Ts complex. Steady-state and dynamic polarization measurements revealed limited local mobility for the tryptophan in the EF-Tu x GDP complex whereas formation of the EF-Tu x EF-Ts complex led to a dramatic increase in this local mobility.     

The affinity of elongation factor Tu for an aminoacyl-tRNA is modulated by the esterified amino acid

When different mutations were introduced into the anticodon loop and at position 73 of YFA2, a derivative of yeast tRNA(Phe), a single tRNA body was misacylated with 13 different amino acids. The affinities of these misacylated tRNAs for Thermus thermophilus elongation factor Tu (EF-Tu).GTP were determined using a ribonuclease protection assay. A range of 2.5 kcal/mol in the binding energies was observed, clearly demonstrating that EF-Tu specifically recognizes the side chain of the esterified amino acid. Furthermore, this specificity can be altered by introducing a mutation in the amino acid binding pocket on the surface of EF-Tu. Also, when discussed in conjunction with the previously determined specificity of EF-Tu for the tRNA body, these experiments further demonstrate that EF-Tu uses thermodynamic compensation to bind cognate aminoacyl-tRNAs similarly.     

Mechanism of elongation factor (EF)-Ts-catalyzed nucleotide exchange in EF-Tu. Contribution of contacts at the guanine base.

Nucleotide exchange in elongation factor Tu (EF-Tu) is catalyzed by elongation factor Ts (EF-Ts). Similarly to other GTP-binding proteins, the structural changes in the P loop and the Mg(2+) binding site are known to be important for nucleotide release from EF-Tu. In the present paper, we determine the contribution of the contacts between helix D of EF-Tu at the base side of the nucleotide and the N-terminal domain of EF-Ts to the catalysis. The rate constants of the multistep reaction between Escherichia coli EF-Tu, EF-Ts, and GDP were determined by stopped-flow kinetic analysis monitoring the fluorescence of either Trp-184 in EF-Tu or mant-GDP. Mutational analysis shows that contacts between helix D of EF-Tu and the N-terminal domain of EF-Ts are important for both complex formation and the acceleration of GDP dissociation. The kinetic results suggest that the initial contact of EF-Ts with helix D of EF-Tu weakens binding interactions around the guanine base, whereas contacts of EF-Ts with the phosphate binding side that promotes the release of the phosphate moiety of GDP appear to take place later. This "base-side-first" mechanism of guanine nucleotide release resembles that found for Ran x RCC1 and differs from mechanisms described for other GTPase x GEF complexes where interactions at the phosphate side of the nucleotide are released first.     

The importance of P-loop and domain movements in EF-Tu for guanine nucleotide exchange

Elongation factor Ts (EF-Ts) is the guanine nucleotide exchange factor for elongation factor Tu (EF-Tu). An important feature of the nucleotide exchange is the structural rearrangement of EF-Tu in the EF-Tu.EF-Ts complex caused by insertion of Phe-81 of EF-Ts between His-84 and His-118 of EF-Tu. In this study, the contribution of His-118 to nucleotide release was studied by pre-steady state kinetic analysis of nucleotide exchange in EF-Tu mutants in which His-118 was replaced by Ala or Glu. Intrinsic as well as EF-Ts-catalyzed release of GDP/GTP was affected by the mutations, resulting in an approximately 10-fold faster spontaneous nucleotide release and a 10-50-fold slower EF-Ts-catalyzed nucleotide release. The effects are attributed to the interference of the mutations with the EF-Ts-induced movements of the P-loop of EF-Tu and changes at the domain 1/3 interface, leading to the release of the beta-phosphate group of GTP/GDP. The K(d) for GTP is increased by more than 40 times when His-118 is replaced with Glu, which may explain the inhibition by His-118 mutations of aminoacyl-tRNA binding to EF-Tu. The mutations had no effect on EF-Tu-dependent delivery of aminoacyl-tRNA to the ribosome.     

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.