lysyl-tRNA Synthetase Research Articles

Phylogeny and shared conserved inserts in proteins provide evidence that Verrucomicrobia are the closest known free-living relatives of chlamydiae

The evolutionary relationships of Chlamydiales, Verrucomicrobia and Planctomycetes were studied based on phylogenetic trees for a concatenated dataset of 11 widely distributed proteins, as well as conserved inserts in several proteins. In phylogenetic trees, a close relationship of chlamydiae to Verrucomicrobium was supported by different phylogenetic methods. Although the Planctomycetes branched close to the chlamydiae-Verrucomicrobia clade, their specific affiliation to these groups was generally not supported. Results are also presented for two conserved inserts, a 6 aa insert in the lysyl-tRNA synthetase and a 3 aa insert in the RNA polymerase beta subunit (RpoB), that are uniquely shared by Verrucomicrobium spinosum and all available Chlamydiales homologues, but which are not found in any of the available Planctomycetes or other bacterial homologues. Signature sequences in a number of other proteins [including a large insert (>150 aa) in DNA gyrase B] provide information regarding the branching position of these groups relative to other bacterial phyla. A close and specific relationship of V. spinosum to the Chlamydiales species, seen both in phylogenetic trees and by means of uniquely shared inserts in protein sequences, strongly indicates that these two groups of species shared a common ancestor exclusive of all other known bacteria. These results suggest that Verrucomicrobia may be the closest free-living relatives of the parasitic chlamydiae.

Human tryptophanyl-tRNA synthetase is switched to a tRNA-dependent mode for tryptophan activation by mutations at V85 and I311

For most aminoacyl-tRNA synthetases (aaRS), their cognate tRNA is not obligatory to catalyze amino acid activation, with the exception of four class I (aaRS): arginyl-tRNA synthetase, glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase and class I lysyl-tRNA synthetase. Furthermore, for arginyl-, glutamyl- and glutaminyl-tRNA synthetase, the integrated 3' end of the tRNA is necessary to activate the ATP-PPi exchange reaction. Tryptophanyl-tRNA synthetase is a class I aaRS that catalyzes tryptophan activation in the absence of its cognate tRNA. Here we describe mutations located at the appended β1–β2 hairpin and the AIDQ sequence of human tryptophanyl-tRNA synthetase that switch this enzyme to a tRNA-dependent mode in the tryptophan activation step. For some mutant enzymes, ATP-PPi exchange activity was completely lacking in the absence of tRNATrp, which could be partially rescued by adding tRNATrp, even if it had been oxidized by sodium periodate. Therefore, these mutant enzymes have strong similarity to arginyl-tRNA synthetase, glutaminyl-tRNA synthetase and glutamyl-tRNA synthetase in their mode of amino acid activation. The results suggest that an aaRS that does not normally require tRNA for amino acid activation can be switched to a tRNA-dependent mode.

Partially Folded States of Staphylococcal Nuclease Highlight the Conserved Structural Hierarchy of OB-Fold Proteins

We have been interested in whether three proteins that share a five-stranded beta-barrel "OB-fold" structural motif but no detectable sequence homology fold by similar mechanisms. Here we describe native-state hydrogen exchange experiments as a function of urea for SN (staphylococcal nuclease), a protein with an OB-fold motif and additional nonconserved elements of structure. The regions of structure with the largest stability and unfolding cooperativity are contained within the conserved OB-fold portion of SN, consistent with previous results for CspA (cold shock protein A) and LysN (anticodon binding domain of lysyl tRNA synthetase). The OB-fold also has the subset of residues with the slowest unfolding rates in the three proteins, as determined by hydrogen exchange experiments in the EX1 limit. Although the protein folding hierarchy is maintained at the level of supersecondary structure, it is not evident for individual residues as might be expected if folding depended on obligatory nucleation sites. Rather, the site-specific stability profiles appear to be linked to sequence hydrophobicity and to the density of long-range contacts at each site in the three-dimensional structures of the proteins. We discuss the implications of the correlation between stability to unfolding and conservation of structure for mechanisms of protein structure evolution.

Role of HIV-1 Vpr-induced apoptosis on the release of mitochondrial lysyl-tRNA synthetase

Mitochondrial lysyl-tRNA synthetase (LysRS) is thought to be involved in the specific packaging of tRNA 3 Lys into HIV-1 viral particles. The HIV-1 auxiliary viral protein Vpr is an apoptogenic protein that affects the integrity of the mitochondrial membrane and has also been reported to interact with LysRS. In the present study, we show that HIV-1 Vpr expressed in E. coli and purified to homogeneity does not interact specifically with LysRS and does not impact its aminoacylation activity. However, we also show that the mitochondrial localization of LysRS in HeLa cells is altered after addition of Vpr in the culture medium. These results suggest that HIV-1 Vpr fulfills an essential role in the process of packaging of mitochondrial LysRS.

Evidence for an Adaptation Mechanism of Mitochondrial Translation via tRNA Import from the Cytosol

Although mitochondrial import of nuclear DNA-encoded RNAs is widely occurring, their functions in the organelles are not always understood. Mitochondrial function(s) of tRNA(Lys)(CUU), tRK1, targeted into Saccharomyces cerevisiae mitochondria was mysterious, since mitochondrial DNA-encoded tRNA(Lys)(UUU), tRK3, was hypothesized to decode both lysine codons, AAA and AAG. Mitochondrial targeting of tRK1 depends on the precursor of mitochondrial lysyl-tRNA synthetase, pre-Msk1p. Here we show that substitution of pre-Msk1p by its Ashbya gossypii ortholog results in a strain in which tRK3 is aminoacylated, while tRK1 is not imported. At elevated temperature, drop of tRK1 import inhibits mitochondrial translation of mRNAs containing AAG codons, which coincides with the impaired 2-thiolation of tRK3 anticodon wobble nucleotide. Restoration of tRK1 import cures the translational defect, suggesting the role of tRK1 in conditional adaptation of mitochondrial protein synthesis. In contrast with the known ways of organellar translation control, this mechanism exploits the RNA import pathway.

The ins and outs of tRNA transport

Transfer RNAs (tRNAs) are involved in many cellular functions distributed throughout the cellular space. In addition to their role in protein translation, these molecules are engaged in tasks as diverse as the regulation of gene expression, amino‐acid synthesis, protein degradation, cell‐wall synthesis, porphyrin biosynthesis, priming of replication, RNA interference and the transport of macromolecules. The nucleus and the mitochondria of eukaryotic cells, as well as the chloroplasts in plants, contain their own genome that encodes a range of proteins and nucleic acids. Early evidence suggested that some of the RNA content of mitochondria could be transcribed from non‐mitochondrial DNA (Suyama, 1967). It is now well established that a variable number of tRNA species present in the mitochondria are indeed nucleus‐encoded (Fig 1). The phenomenon of mitochondrial tRNA import has been reported in plants, marsupials, the yeast Saccharomyces cerevisiae , and the protozoa Leishmania , Trypanosoma and Tetrahymena . When certain tRNA species are not encoded by the mitochondrial genome, there is a predictable requirement for the import of nucleus‐encoded tRNAs into the mitochondria (Schneider & Marechal‐Drouard, 2000). This is the case, for example, for the mitochondria of Leishmania , which are completely devoid of tRNA‐encoding genes. However, there are a few examples of tRNA import into mitochondria that already have a complete set of mitochondria‐encoded tRNAs. Other tRNA transport systems have also been identified (Fig 1), which include their retrograde transport from the cytoplasm to the nucleus (Takano et al , 2005; Shaheen & Hopper, 2005), the export of mitochondria‐encoded tRNA to the cytoplasm (Maniataki & Mourelatos, 2005), and the packaging of tRNAs into retroviruses (Waters & Mullin, 1977). The roles of some of these transport systems have yet to be defined. Figure 1. The travels of transfer RNA. …

Class I and class II lysyl‐tRNA synthetase mutants and the genetic encoding of pyrrolysine in Methanosarcina spp.

Methanosarcina spp. begin methanogenesis from methylamines with methyltransferases made via the translation of UAG as pyrrolysine. In vitro evidence indicates two possible routes to pyrrolysyl-tRNA(Pyl). PylS ligates pyrrolysine to tRNA(Pyl). Alternatively, class I and class II lysyl-tRNA synthetases (LysRS1 and LysRS2) together form lysyl-tRNA(Pyl), a potential intermediate to pyrrolysyl-tRNA(Pyl). The unusual possession of both LysRS1 and LysRS2 by Methanosarcina spp. may also reflect differences in catalytic properties. Here we assessed the in vivo relevance of these hypotheses. The lysK and mtmB transcripts, encoding LysRS1 and monomethylamine methyltransferase, were detectable in Methanosarcina barkeri during early log growth on trimethylamine, but not methanol. In contrast, lysS transcript encoding LysRS2 was detectable during log phase with either substrate. Methanosarcina acetivorans strains bearing deletions of lysK or lysS grew normally on methanol and methylamines with wild-type levels of monomethylamine methyltransferase and aminoacyl-tRNA(Pyl). The lysK and lysS genes could not replace pylS in a recombinant system employing tRNA(Pyl) for UAG suppression. The results support an association of LysRS1 with growth on methylamine, but not an essential role for LysRS1/LysRS2 in the genetic encoding of pyrrolysine. However, decreased lysyl-tRNA(Lys) in the lysS mutant provides a possible rationale for stable transfer of the bacterial lysS gene to methanoarchaea.

Engineered Monomeric Human Histidine Triad Nucleotide-binding Protein 1 Hydrolyzes Fluorogenic Acyl-adenylate and Lysyl-tRNA Synthetase-generated Lysyl-adenylate

Hint1 is a homodimeric protein and member of the ubiquitous HIT superfamily. Hint1 catalyzes the hydrolysis of purine phosphoramidates and lysyl-adenylate generated by lysyl-tRNA synthetase (LysRS). To determine the importance of homodimerization on the biological and catalytic activity of Hint1, the dimer interface of human Hint1 (hHint1) was destabilized by replacement of Val(97) of hHint1 with Asp, Glu, or Arg. The mutants were shown to exist as monomers in solution by a combination of size exclusion chromatograph, static light scattering, and chemically induced dimerization studies. Circular dichroism studies revealed little difference between the stability of the V97D, V97E, and wild-type hHint1. Relative to wild-type and the V97E mutant, however, significant perturbation of the V97D mutant structure was observed. hHint1 was shown to prefer 3-indolepropionic acyl-adenylate (AIPA) over tryptamine adenosine phosphoramidate monoester (TpAd). Wild-type hHint1 was found to be 277- and 1000-fold more efficient (k(cat)/K(m) values) than the V97E and V97D mutants, respectively. Adenylation of wild-type, V97D, and V97E hHint1 by human LysRS was shown to correlate with the mutant k(cat)/K(m) values using 3-indolepropionic acyl-adenylate as a substrate, but not tryptamine adenosine phosphoramidate monoester. Significant perturbations of the active site residues were not detected by molecular dynamics simulations of the hHint1s. Taken together, these results demonstrate that for hHint1; 1) the efficiency (k(cat)/K(m)) of acylated AMP hydrolysis, but not maximal catalytic turnover (k(cat)), is dependent on homodimerization and 2) the hydrolysis of lysyl-AMP generated by LysRS is not dependent on homodimerization if the monomer structure is similar to the wild-type structure.

Altered expression of plant lysyl tRNA synthetase promotes tRNA misacylation and translational recoding of lysine

The Arabidopsis thaliana lysyl tRNA synthetase (AtKRS) structurally and functionally resembles the well-characterized prokaryotic class IIb KRS, including the propensity to aminoacylate tRNA(Lys) with suboptimal identity elements, as well as non-cognate tRNAs. Transient expression of AtKRS in carrot cells promotes aminoacylation of such tRNAs in vivo and translational recoding of lysine at nonsense codons. Stable expression of AtKRS in Zea mays causes translational recoding of lysine into zeins, significantly enriching the lysine content of grain.

N‐terminal domains of native multidomain proteins have the potential to assist de novo folding of their downstream domains in vivo by acting as solubility enhancers

The fusion of soluble partner to the N terminus of aggregation-prone polypeptide has been popularly used to overcome the formation of inclusion bodies in the E. coli cytosol. The chaperone-like functions of the upstream fusion partner in the artificial multidomain proteins could occur in de novo folding of native multidomain proteins. Here, we show that the N-terminal domains of three E. coli multidomain proteins such as lysyl-tRNA synthetase, threonyl-tRNA synthetase, and aconitase are potent solubility enhancers for various C-terminal heterologous proteins. The results suggest that the N-terminal domains could act as solubility enhancers for the folding of their authentic C-terminal domains in vivo. Tandem repeat of N-terminal domain or insertion of aspartic residues at the C terminus of the N-terminal domain also increased the solubility of fusion proteins, suggesting that the solubilizing ability correlates with the size and charge of N-terminal domains. The solubilizing ability of N-terminal domains would contribute to the autonomous folding of multidomain proteins in vivo, and based on these results, we propose a model of how N-terminal domains solubilize their downstream domains.

Lysyl-tRNA Synthetase-generated Lysyl-Adenylate Is a Substrate for Histidine Triad Nucleotide Binding Proteins

Histidine triad nucleotide binding proteins (Hints) are the most ancient members of the histidine triad protein superfamily of nucleotidyltransferases and hydrolyases. Protein-protein interaction studies have found that complexes of the transcription factors MITF or USF2 and lysyl-tRNA synthetase (LysRS) are associated with human Hint1. Therefore, we hypothesized that lysyl-AMP or the LysRS.lysyl-AMP may be a native substrate for Hints. To explore the biochemical relationship between Hint1 and LysRS, a series of catalytic radiolabeling, mutagenesis, and kinetic experiments was conducted with purified LysRSs and Hints from human and Escherichia coli. After incubation of the E. coli or human LysRS with Hints and [alpha-(32)P]ATP, but not [alpha-(32)P]GTP, (32)P-labeled Hints were observed. By varying time and the concentrations of lysine, Mg(2+), or LysRS, the adenylation of Hint was found to be dependent on the formation of lysyl-AMP. Site-directed mutagenesis studies of the active site histidine triad revealed that Hint labeling could be abolished by substitution of either His-101 of E. coli hinT or His-112 of human Hint1 by either alanine or glycine. Ap(4)A, believed to be synthesized by LysRS in vivo, and Zn(2+) were shown to inhibit the formation of Hint-AMP with an IC(50) value in the low micromolar range. Consistent with pyrophosphate being an inhibitor for aminoacyl-tRNA synthetase, incubations in the presence of pyrophosphatase resulted in enhanced formation of Hint-AMP. These results demonstrate that the lysyl-AMP intermediate formed by LysRS is a natural substrate for Hints and suggests a potential highly conserved regulatory role for Hints on LysRS and possibly other aminoacyl-tRNA synthetases.

Functional Association between Three Archaeal Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching amino acids to their cognate tRNAs during protein synthesis. In eukaryotes aaRSs are commonly found in multi-enzyme complexes, although the role of these complexes is still not completely clear. Associations between aaRSs have also been reported in archaea, including a complex between prolyl-(ProRS) and leucyl-tRNA synthetases (LeuRS) in Methanothermobacter thermautotrophicus that enhances tRNA(Pro) aminoacylation. Yeast two-hybrid screens suggested that lysyl-tRNA synthetase (LysRS) also associates with LeuRS in M. thermautotrophicus. Co-purification experiments confirmed that LeuRS, LysRS, and ProRS associate in cell-free extracts. LeuRS bound LysRS and ProRS with a comparable K(D) of about 0.3-0.9 microm, further supporting the formation of a stable multi-synthetase complex. The steady-state kinetics of aminoacylation by LysRS indicated that LeuRS specifically reduced the Km for tRNA(Lys) over 3-fold, with no additional change seen upon the addition of ProRS. No significant changes in aminoacylation by LeuRS or ProRS were observed upon the addition of LysRS. These findings, together with earlier data, indicate the existence of a functional complex of three aminoacyl-tRNA synthetases in archaea in which LeuRS improves the catalytic efficiency of tRNA aminoacylation by both LysRS and ProRS.

Brain proteins interacting with the tetramerization region of non-erythroid alpha spectrin

The N-terminal region of non-erythroid alpha spectrin (SpαII) is responsible for interacting with its binding partner, beta spectrin, to form functional spectrin tetramers. We used a yeast-two-hybrid system, with an N-terminal segment of alpha spectrin representing the functional tetramerization site, as a bait to screen human brain c-DNA library for proteins that interact with the alpha spectrin segment. In addition to several beta spectrin isoforms, we identified 14 proteins that interact with SpαII. Seven of the 14 were matched to 6 known proteins: Duo protein, Lysyl-tRNA synthetase, TBP associated factor 1, two isoforms (b and c) of a protein kinase A interacting protein and Zinc finger protein 333 (2 different segments). Four of the 6 proteins are located primarily in the nucleus, suggesting that spectrin plays important roles in nuclear functions. The remaining 7 proteins were unknown to the protein data base. Structural predictions show that many of the 14 proteins consist of a large portion of unstructured regions, suggesting that many of these proteins fold into a rather flexible conformation. It is interesting to note that all but 3 of the 14 proteins are predicted to consist of one to four coiled coils (amphiphilic helices). A mutation in SpαII, V22D, which interferes with the coiled coil bundling of SpαII with beta spectrin, also affects SpαII interaction with Duo protein, TBP associated factor 1 and Lysyl-tRNA synthetase, suggesting that they may compete with beta spectrin for interaction with SpαII. Future structural and functional studies of these proteins to provide interaction mechanisms will no doubt lead to a better understanding of brain physiology and pathophysiology.

Hierarchical Network between the Components of the Multi-tRNA Synthetase Complex

The macromolecular tRNA synthetase complex consists of nine different enzymes and three non-enzymatic factors. This complex was recently shown to be a novel signalosome, since many of its components are involved in signaling pathways in addition to their catalytic roles in protein synthesis. The structural organization and dynamic relationships of the components of the complex are not well understood. Here we performed a systematic depletion analysis to determine the effects of structural intimacy and the turnover of the components. The results showed that the stability of some components depended on their neighbors. Lysyl-tRNA synthetase was most independent of other components for its stability whereas it was most required for the stability of other components. Arginyl- and methionyl-tRNA synthetases had the opposite characteristics. Thus, the systematic depletion of the components revealed the functional reason for the complex formation and the assembly pattern of these multi-functional enzymes and their associated factors.

Viral Hijacking of Mitochondrial Lysyl-tRNA Synthetase

The primer for reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome is tRNA3(Lys). During assembly of HIV-1 particles, tRNA3(Lys) is taken up from the host cell along with lysyl-tRNA synthetase (LysRS), the tRNA binding protein that specifically aminoacylates the different tRNA(Lys) isoacceptors. In humans, the cytoplasmic and mitochondrial species of LysRS are encoded by a single gene by means of alternative splicing. Here, we show that polyclonal antibodies directed to the full-length cytoplasmic enzyme equally recognized the two enzyme species. We raised antibodies against synthetic peptides that allowed discrimination between the two enzymes and found that mitochondrial LysRS is the only cellular source of LysRS detected in the virions. These results open new routes for understanding the molecular mechanisms involved in the specific packaging of tRNA3(Lys) into viral particles.

Lysyl-tRNA synthetase: A target for mutant SOD1 toxicity in mitochondria

Lysyl-tRNA synthetase: A target for mutant SOD1 toxicity in mitochondria

Complementation of Human Immunodeficiency Virus Type 1 Replication by Intracellular Selection of Escherichia coli \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} Supplied in trans

Human immunodeficiency virus type 1 (HIV-1) exclusively selects tRNA3Lys as the primer for the initiation of reverse transcription, even though both tRNA3Lys and tRNA1,2Lys are found in HIV-1 virions. Alteration of the HIV-1 primer-binding site (PBS) to be complementary to alternate tRNAs results in the use of those tRNAs for replication, indicating that primer complementarity with the PBS is an important determinant of primer selection. In previous studies, we have exploited this fact to develop a system in which yeast (Saccharomyces cerevisiae) tRNAPhe is provided in trans to complement the replication of HIV-1 with a PBS complementary to yeast tRNAPhe. Recent studies have demonstrated that the presence of lysyl-tRNA synthetase in HIV-1 virions might account for the preference for the selection of tRNA3Lys in HIV-1 replication. To establish a complementation system more reflective of HIV-1 primer selection, we have altered the HIV-1 PBS to be complementary to the Escherichia coli tRNA3Lys, which shares near identity with mammalian tRNA3Lys except in the 3'-terminal 18-nucleotide sequence that binds to the PBS. E. coli tRNA3Lys expressed from a plasmid was aminoacylated in mammalian cells. Cotransfection of cells with a plasmid that encodes E. coli tRNA3Lys and a plasmid encoding an HIV-1 provirus with a PBS complementary to E. coli tRNA3Lys resulted in the production of infectious virus. A comparison of the two complementation systems revealed that higher levels of intracellular E. coli tRNA3Lys than of yeast tRNAPhe were needed to achieve equal levels of infectious virus, indicating that there was no preferential selection of E. coli tRNA3Lys. To examine the specificity of tRNALys selection, E. coli tRNA3Lys was modified to tRNA1,2Lys. This tRNA was also aminoacylated when expressed in mammalian cells and complemented the infectivity of HIV-1 at levels similar to those seen for E. coli tRNA3Lys. Additional mutations in the anticodon of E. coli tRNA3Lys were constructed; these mutations did not significantly correlate with the capacity of the tRNA primer to complement infectivity of HIV-1, even though they had a drastic effect on the aminoacylation of the tRNAs. The results of these studies demonstrate that E. coli tRNA3Lys provided in trans can complement HIV-1 genomes with the PBS altered to E. coli tRNA3Lys. However, the capacity of tRNA3Lys to interact with lysyl-tRNA synthetase does not entirely explain the enhanced preference for selection of tRNA3Lys for the replication of HIV-1.

Design of a Bacterial Host for Site-Specific Incorporation of p-Bromophenylalanine into Recombinant Proteins

Introduction of a yeast suppressor tRNA (ytRNA(Phe)(CUA)) and a mutant yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) into an Escherichia coli expression host allows in vivo incorporation of phenylalanine analogues into recombinant proteins in response to amber stop codons. However, high-fidelity incorporation of non-natural amino acids is precluded in this system by mischarging of ytRNA(Phe)(CUA) with tryptophan (Trp) and lysine (Lys). Here we show that ytRNA(Phe)(CUA) and yPheRS can be redesigned to achieve high-fidelity amber codon suppression through delivery of p-bromophenylalanine (pBrF). Two strategies were used to reduce misincorporation of Trp and Lys. First, Lys misincorporation was eliminated by disruption of a Watson-Crick base pair between nucleotides 30 and 40 in ytRNA(Phe)(CUA). Loss of this base pair reduces mischarging by the E. coli lysyl-tRNA synthetase. Second, the binding site of yPheRS was redesigned to enhance specificity for pBrF. Specifically, we used the T415A variant, which exhibits 5-fold higher activity toward pBrF as compared to Trp in ATP-PP(i) exchange assays. Combining mutant ytRNA(Phe)(CUA) and yPheRS (T415A) allowed incorporation of pBrF into murine dihydrofolate reductase in response to an amber codon with at least 98% fidelity.

Identity Elements for Specific Aminoacylation of a tRNA by Mammalian Lysyl-tRNA Synthetase Bearing a Nonspecific tRNA-Interacting Factor

Mammalian lysyl-tRNA synthetase (LysRS) has an N-terminal polypeptide chain extension appended to a prokaryotic-like synthetase domain. This extension, termed a tRNA-interacting factor (tIF), possesses a RNA-binding motif [KxxxK(K/R)xxK] that binds nonspecifically the acceptor TPsiC stem-loop domain of tRNA and provides a potent tRNA binding capacity to this enzyme. Consequently, native LysRS aminoacylates a RNA minihelix mimicking the amino acid acceptor stem-loop domain of tRNA(3)(Lys). Here, examination of minihelix recognition showed that mammalian LysRS aminoacylates RNA minihelices without specificity of sequence, revealing that none of the nucleotides from the acceptor TPsiC stem-loop domain are essential determinants of tRNA(Lys) acceptor identity. To test whether the tIF domain reduces the specificity of the synthetase with regard to complete tRNA molecules, aminoacylation of wild-type and mutant noncognate tRNAs by wild-type or N-terminally truncated LysRS was examined. The presence of the UUU anticodon of tRNA(Lys) appeared to be necessary and sufficient to transform yeast tRNA(Asp) or tRNA(i)(Met) into potent lysine acceptor tRNAs. Thus, nonspecific RNA-protein interactions between the acceptor stem of tRNA and the tIF domain do not relax the tRNA specificity of mammalian LysRS. The possibility that interaction of the full-length cognate tRNA with the synthetase is required to induce the catalytic center of the enzyme into a productive conformation is discussed.

The duality of LysU, a catalyst for both Ap4A and Ap3A formation

Heat shock inducible lysyl-tRNA synthetase of Escherichia coli (LysU) is known to be a highly efficient diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) synthase. However, we use an ion-exchange HPLC technique to demonstrate that active LysU mixtures actually have a dual catalytic activity, initially producing Ap4A from ATP, before converting that tetraphosphate to a triphosphate. LysU appears to be an effective diadenosine 5',5'''-P1,P3-triphosphate (Ap3A) synthase. Mechanistic investigations reveal that Ap3A formation requires: (a) that the second step of Ap4A formation is slightly reversible, thereby leading to a modest reappearance of adenylate intermediate; and (b) that phosphate is present to trap the intermediate (either as inorganic phosphate, as added ADP, or as ADP generated in situ from inorganic phosphate). Ap3A forms readily from Ap4A in the presence of such phosphate-based adenylate traps (via a 'reverse-trap' mechanism). LysU is also clearly demonstrated to exist in a phosphorylated state that is more physically robust as a catalyst of Ap4A formation than the nonphosphorylated state. However, phosphorylated LysU shows only marginally improved catalytic efficiency. We note that Ap3A effects have barely been studied in prokaryotic organisms. By contrast, there is a body of literature that describes Ap3A and Ap4A having substantially different functions in eukaryotic cells. Our data suggest that Ap3A and Ap4A biosynthesis could be linked together through a single prokaryotic dual 'synthase' enzyme. Therefore, in our view there is a need for new research into the effects and impact of Ap3A alone and the intracellular [Ap3A]/[Ap4A] ratio on prokaryotic organisms.