Misacylation of tRNA with methionine in Saccharomyces cerevisiae

Accurate transfer RNA (tRNA) aminoacylation by aminoacyl-tRNA synthetases controls translational fidelity. Although tRNA synthetases are generally highly accurate, recent results show that the methionyl-tRNA synthetase (MetRS) is an exception. MetRS readily misacylates non-methionyl tRNAs at frequencies of up to 10% in mammalian cells; such mismethionylation may serve a beneficial role for cells to protect their own proteins against oxidative damage. The Escherichia coli MetRS mismethionylates two E. coli tRNA species in vitro, and these two tRNAs contain identity elements for mismethionylation. Here we investigate tRNA mismethionylation in Saccharomyces cerevisiae. tRNA mismethionylation occurs at a similar extent in vivo as in mammalian cells. Both cognate and mismethionylated tRNAs have similar turnover kinetics upon cycloheximide treatment. We identify specific arginine/lysine to methionine-substituted peptides in proteomic mass spectrometry, indicating that mismethionylated tRNAs are used in translation. The yeast MetRS is part of a complex containing the anchoring protein Arc1p and the glutamyl-tRNA synthetase (GluRS). The recombinant Arc1p–MetRS–GluRS complex binds and mismethionylates many tRNA species in vitro. Our results indicate that the yeast MetRS is responsible for extensive misacylation of non-methionyl tRNAs, and mismethionylation also occurs in this evolutionary branch.     

A relationship between asparagine synthetase A and aspartyl tRNA synthetase.

A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A. The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase. To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases. The resulting genes were expressed in E. coli, and the gene products were assayed for asparagine synthetase activity in vitro. Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity. Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298. Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity. These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E. coli, evolved from an ancestral aminoacyl tRNA synthetase.     

Competition of aminoacyl-tRNA synthetases for tRNA ensures the accuracy of aminoacylation

The accuracy of protein biosynthesis rests on the high fidelity with which aminoacyl-tRNA synthetases discriminate between tRNAs. Correct aminoacylation depends not only on identity elements (nucleotides in certain positions) in tRNA (1), but also on competition between different synthetases for a given tRNA (2). Here we describe in vivo and in vitro experiments which demonstrate how variations in the levels of synthetases and tRNA affect the accuracy of aminoacylation. We show in vivo that concurrent overexpression of Escherichia coli tyrosyl-tRNA synthetase abolishes misacylation of supF tRNA^Tyr with glutamine in vivo by overproduced glutaminyl-tRNA synthetase. In an in vitro competition assay, we have confirmed that the overproduction mischarging phenomenon observed in vivo is due to competition between the synthetases at the level of aminoacylation. Likewise, we have been able to examine the role competition plays in the identity of a non-suppressor tRNA of ambiguous identity, tRNA^Glu. Finally, with this assay, we show that the identity of a tRNA and the accuracy with which it is recognized depend on the relative affinities of the synthetases for the tRNA. The in vitro competition assay represents a general method of obtaining qualitative information on tRNA identity in a competitive environment (usually only found in vivo) during a defined step in protein biosynthesis, aminoacylation. In addition, we show that the discriminator base (position 73) and the first base of the anticodon are important for recognition by E. coli tyrosyl-tRNA synthetase.     

Lyme disease transmission by severely impaired ticks

It has been demonstrated that impairing protein synthesis using drugs targeted against tRNA amino acid synthetases presents a promising strategy for the treatment of a wide variety of parasitic diseases, including malaria and toxoplasmosis. This is the first study evaluating tRNA synthetases as potential drug targets in ticks. RNAi knock-down of all tested tRNA synthetases had a strong deleterious phenotype on Ixodes ricinus feeding. Our data indicate that tRNA synthetases represent attractive, anti-tick targets warranting the design of selective inhibitors. Further, we tested whether these severely impaired ticks were capable of transmitting Borrelia afzelii spirochaetes. Interestingly, biologically handicapped I. ricinus nymphs transmitted B. afzelii in a manner quantitatively sufficient to develop a systemic infection in mice. These data suggest that initial blood-feeding, despite the incapability of ticks to fully feed and salivate, is sufficient for activating B. afzelii from a dormant to an infectious mode, enabling transmission and dissemination in host tissues.     

Essentiality Assessment of Cysteinyl and Lysyl-tRNA Synthetases of Mycobacterium smegmatis

Discovery of mupirocin, an antibiotic that targets isoleucyl-tRNA synthetase, established aminoacyl-tRNA synthetase as an attractive target for the discovery of novel antibacterial agents. Despite a high degree of similarity between the bacterial and human aminoacyl-tRNA synthetases, the selectivity observed with mupirocin triggered the possibility of targeting other aminoacyl-tRNA synthetases as potential drug targets. These enzymes catalyse the condensation of a specific amino acid to its cognate tRNA in an energy-dependent reaction. Therefore, each organism is expected to encode at least twenty aminoacyl-tRNA synthetases, one for each amino acid. However, a bioinformatics search for genes encoding aminoacyl-tRNA synthetases from Mycobacterium smegmatis returned multiple genes for glutamyl (GluRS), cysteinyl (CysRS), prolyl (ProRS) and lysyl (LysRS) tRNA synthetases. The pathogenic mycobacteria, namely, Mycobacterium tuberculosis and Mycobacterium leprae, were also found to possess two genes each for CysRS and LysRS. A similar search indicated the presence of additional genes for LysRS in gram negative bacteria as well. Herein, we describe sequence and structural analysis of the additional aminoacyl-tRNA synthetase genes found in M. smegmatis. Characterization of conditional expression strains of Cysteinyl and Lysyl-tRNA synthetases generated in M. smegmatis revealed that the canonical aminoacyl-tRNA synthetase are essential, while the additional ones are not essential for the growth of M. smegmatis.     

Aminoacylation of RNA Minihelices: Implications for tRNA Synthetase Structural Design and Evolution

Abstract

The genetic code is based on the aminoacylation of tRNA with amino acids catalyzed by the aminoacyl-tRNA synthetases. The synthetases are constructed from discrete domains and all synthetases possess a core catalytic domain that catalyzes amino acid activation, binds the acceptor stem of tRNA, and transfers the amino acid to tRNA. Fused to the core domain are additional domains that mediate RNA interactions distal to the acceptor stem. Several synthetases catalyze the aminoacylation of RNA oligonucleotide substrates that recreate only the tRNA acceptor stems. In one case, a relatively small catalytic domain catalyzes the aminoacylation of these substrates independent of the rest of the protein. Thus, the active site domain may represent a primordial synthetase in which polypeptide insertions that mediate RNA acceptor stem interactions are tightly integrated with determinants for aminoacyl adenylate synthesis. The relationship between nucleotide sequences in small RNA oligonucleotides and the specific amino acids that are attached to these oligonucleotides could constitute a second genetic code.     

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.     

Structural analyses of the malaria parasite aminoacyl‐tRNA synthetases provide new avenues for antimalarial drug discovery

Malaria is a parasitic illness caused by the genus Plasmodium from the apicomplexan phylum. Five plasmodial species of P. falciparum (Pf), P. knowlesi, P. malariae, P. ovale, and P. vivax (Pv) are responsible for causing malaria in humans. According to the World Malaria Report 2020, there were 229 million cases and ~ 0.04 million deaths of which 67% were in children below 5 years of age. While more than 3 billion people are at risk of malaria infection globally, antimalarial drugs are their only option for treatment. Antimalarial drug resistance keeps arising periodically and thus threatens the main line of malaria treatment, emphasizing the need to find new alternatives. The availability of whole genomes of P. falciparum and P. vivax has allowed targeting their unexplored plasmodial enzymes for inhibitor development with a focus on multistage targets that are crucial for parasite viability in both the blood and liver stages. Over the past decades, aminoacyl‐tRNA synthetases (aaRSs) have been explored as anti‐bacterial and anti‐fungal drug targets, and more recently (since 2009) aaRSs are also the focus of antimalarial drug targeting. Here, we dissect the structure‐based knowledge of the most advanced three aaRSs—lysyl‐ (KRS), prolyl‐ (PRS), and phenylalanyl‐ (FRS) synthetases in terms of development of antimalarial drugs. These examples showcase the promising potential of this family of enzymes to provide druggable targets that stall protein synthesis upon inhibition and thereby kill malaria parasites selectively.     

Anticodon recognition in evolution: switching tRNA specificity of an aminoacyl-tRNA synthetase by site-directed peptide transplantation

The highly conserved aspartyl-, asparaginyl-, and lysyl-tRNA synthetases compose one subclass of aminoacyl-tRNA synthetases, called IIb. The three enzymes possess an OB-folded extension at their N terminus. The function of this extension is to specifically recognize the anticodon triplet of the tRNA. Three-dimensional models of bacterial aspartyl- and lysyl-tRNA synthetases complexed to tRNA indicate that a rigid scaffold of amino acid residues along the five beta-strands of the OB-fold accommodates the base U at the center of the anticodon. The binding of the adjacent anticodon bases occurs through interactions with a flexible loop joining strands 4 and 5 (L45). As a result, a switching of the specificity of lysyl-tRNA synthetase from tRNALys (anticodon UUU) toward tRNAAsp (GUC) could be attempted by transplanting the small loop L45 of aspartyl-tRNA synthetase inside lysyl-tRNA synthetase. Upon this transplantation, lysyl-tRNA synthetase loses its capacity to aminoacylate tRNALys. In exchange, the chimeric enzyme acquires the capacity to charge tRNAAsp with lysine. Upon giving the tRNAAsp substrate the discriminator base of tRNALys, the specificity shift is improved. The change of specificity was also established in vivo. Indeed, the transplanted lysyl-tRNA synthetase succeeds in suppressing a missense Lys --> Asp mutation inserted into the beta-lactamase gene. These results functionally establish that sequence variation in a small peptide region of subclass IIb aminoacyl-tRNA synthetases contributes to specification of nucleic acid recognition. Because this peptide element is not part of the core catalytic structure, it may have evolved independently of the active sites of these synthetases.     

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNA^Arg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure–function understanding in prokaryotic tRNA^Arg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNA^Arg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNA^Arg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed.     

Adaptation of aminoacylation identity rules to mammalian mitochondria

Many mammalian mitochondrial aminoacyl-tRNA synthetases are of bacterial-type and share structural domains with homologous bacterial enzymes of the same specificity. Despite this high similarity, synthetases from bacteria are known for their inability to aminoacylate mitochondrial tRNAs, while mitochondrial enzymes do aminoacylate bacterial tRNAs. Here, the reasons for non-aminoacylation by a bacterial enzyme of a mitochondrial tRNA have been explored. A mutagenic analysis performed on in vitro transcribed human mitochondrial tRNA^Asp variants tested for their ability to become aspartylated by Escherichia coli aspartyl-tRNA synthetase, reveals that full conversion cannot be achieved on the basis of the currently established tRNA/synthetase recognition rules. Integration of the full set of aspartylation identity elements and stabilization of the structural tRNA scaffold by restoration of D- and T-loop interactions, enable only a partial gain in aspartylation efficiency. The sequence context and high structural instability of the mitochondrial tRNA are additional features hindering optimal adaptation of the tRNA to the bacterial enzyme. Our data support the hypothesis that non-aminoacylation of mitochondrial tRNAs by bacterial synthetases is linked to the large sequence and structural relaxation of the organelle encoded tRNAs, itself a consequence of the high rate of mitochondrial genome divergence.     

Molecular evolution of protein-RNA mimicry as a mechanism for translational control

Elongation factor P (EF-P) is a conserved ribosome-binding protein that structurally mimics tRNA to enable the synthesis of peptides containing motifs that otherwise would induce translational stalling, including polyproline. In many bacteria, EF-P function requires post-translational modification with (R)-β-lysine by the lysyl-tRNA synthetase paralog PoxA. To investigate how recognition of EF-P by PoxA evolved from tRNA recognition by aminoacyl-tRNA synthetases, we compared the roles of EF-P/PoxA polar contacts with analogous interactions in a closely related tRNA/synthetase complex. PoxA was found to recognize EF-P solely via identity elements in the acceptor loop, the domain of the protein that interacts with the ribosome peptidyl transferase center and mimics the 3''-acceptor stem of tRNA. Although the EF-P acceptor loop residues required for PoxA recognition are highly conserved, their conservation was found to be independent of the phylogenetic distribution of PoxA. This suggests EF-P first evolved tRNA mimicry to optimize interactions with the ribosome, with PoxA-catalyzed aminoacylation evolving later as a secondary mechanism to further improve ribosome binding and translation control.     

The mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena are indistinguishable.

The mitochondrial and cytoplasmic valyl tRNA synthetases from Tetrahymena pyriformis are indistinguishable. These synthetases cannot be differentiated through hydroxylapatite, DEAE-cellulose, or phosphocellulose column chromatography. Both enzymes show the same mean sedimentation coefficient of 5.9 S in sucrose gradient centrifugation analysis; when bound with tRNA, they are relatively stable and sediment at 7.8 S. The temperature optimum for aminoacylation reaction is 27.5 °C, the optimum Mg²⁺ concentration is 4.4 mm, and substrate affinities (K_m values) for valine and ATP in aminoacylation are the same for both enzymes at 1.0 μm and 2.5 mm, respectively. These enzymes show identical specificities for acylation of different tRNA species, i.e., Tetrahymena and rat liver tRNAs can be equally well recognized, but no significant acylation can be observed with Escherichia coli and Saccharomyces tRNAs. These observations suggest the probable molecular identity of mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena.     

Conservation and function of Rab small GTPases in Entamoeba: Annotation of E. invadens Rab and its use for the understanding of Entamoeba biology

Entamoeba invadens is a reptilian enteric protozoan parasite closely related to the human pathogen Entamoeba histolytica and a good model organism of encystation. To understand the molecular mechanism of vesicular trafficking involved in the encystation of Entamoeba, we examined the conservation of Rab small GTPases between the two species. E. invadens has over 100 Rab genes, similar to E. histolytica. Most of the Rab subfamilies are conserved between the two species, while a number of species-specific Rabs are also present. We annotated all E. invadens Rabs according to the previous nomenclature [Saito-Nakano, Y., Loftus, B.J., Hall, N., Nozaki, T., 2005. The diversity of Rab GTPases in Entamoeba histolytica. Experimental Parasitology 110, 244-252]. Comparative genomic analysis suggested that the fundamental vesicular traffic machinery is well conserved, while there are species-specific protein transport mechanisms. We also reviewed the function of Rabs in Entamoeba, and proposed the use of the annotation of E. invadens Rab genes to understand the ubiquitous importance of Rab-mediated membrane trafficking during important biological processes including differentiation in Entamoeba.     

The Acetylation Motif in AMP-Forming Acyl Coenzyme A Synthetases Contains Residues Critical for Acetylation and Recognition by the Protein Acetyltransferase Pat of Rhodopseudomonas palustris

The AMP-forming acyl coenzyme A (acyl-CoA) synthetases are a large class of enzymes found in both anabolic and catabolic pathways that activate fatty acids to acyl-CoA molecules. The protein acetyltransferase (Pat) from Rhodopseudomonas palustris (RpPat) inactivates AMP-forming acyl-CoA synthetases by acetylating the ε-amino group of a conserved, catalytic lysine residue. In all of the previously described RpPat substrates, this lysine residue is located within a PX₄GK motif that has been proposed to be a recognition motif for RpPat. Here, we report five new substrates for RpPat, all of which are also AMP-forming acyl-CoA synthetases. This finding supports the idea that Pat enzymes may have evolved to control the activity of this family of enzymes. Notably, RpPat did not acetylate the wild-type long-chain acyl-CoA synthetase B (RpLcsB; formerly Rpa2714) enzyme of this bacterium. However, a single amino acid change two residues upstream of the acetylation site was sufficient to convert RpLcsB into an RpPat substrate. The results of mutational and functional analyses of RpLcsB and RpPimA variants led us to propose PK/RTXS/T/V/NGKX₂K/R as a substrate recognition motif. The underlined positions within this motif are particularly important for acetylation by RpPat. The first residue, threonine, is located 4 amino acids upstream of the acetylation site. The second residue can be S/T/V/N and is located two positions upstream of the acetylation site. Analysis of published crystal structures suggests that the side chains of these two residues are very close to the acetylated lysine residue, indicating that they may directly interact with RpPat.     

Non-essential role of lysine residues for the catalytic activities of aspartyl-tRNA synthetase and comparison with other aminoacyl-tRNA synthetases

Essential lysine residues were sought in the catalytic site of baker's yeast aspartyl-tRNA synthetase (an alpha 2 dimer of Mr 125,000) using affinity labeling methods and periodate-oxidized adenosine, ATP, and tRNA(Asp). It is shown that the number of periodate-oxidized derivatives which can be bound to the synthetase via Schiff's base formation with epsilon-NH2 groups of lysine residues exceeds the stoichiometry of specific substrate binding. Furthermore, it is found that the enzymatic activities are not completely abolished, even for high incorporation levels of the modified substrates. The tRNA(Asp) aminoacylation reaction is more sensitive to labeling than is the ATP-PPi exchange one; for enzyme preparations modified with oxidized adenosine or ATP this activity remains unaltered. These results demonstrate the absence of a specific lysine residue directly involved in the catalytic activities of yeast aspartyl-tRNA synthetase. Comparative labeling experiments with oxidized ATP were run with several other aminoacyl-tRNA synthetases. Residual ATP-PPi exchange and tRNA aminoacylation activities measured in each case on the modified synthetases reveal different behaviors of these enzymes when compared to that of aspartyl-tRNA synthetase. When tested under identical experimental conditions, pure isoleucyl-, methionyl-, threonyl- and valyl-tRNA synthetases from E. coli can be completely inactivated for their catalytic activities; for E. coli alanyl-tRNA synthetase only the tRNA charging activity is affected, whereas yeast valyl-tRNA synthetase is only partly inactivated. The structural significance of these experiments and the occurrence of essential lysine residues in aminoacyl-tRNA synthetases are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulated Capture by Exosomes of mRNAs for Cytoplasmic tRNA Synthetases

Although tRNA synthetases are enzymes that catalyze the first step of translation in the cytoplasm, surprising functions unrelated to translation have been reported. These studies, and the demonstration of novel activities of splice variants, suggest a far broader reach of tRNA synthetases into cell biology than previously recognized. Here we show that mRNAs for most tRNA synthetases can be detected in exosomes. Also detected in exosomes was an mRNA encoding a unique splice variant that others had associated with prostate cancer. The exosomal mRNAs encoding the native synthetase and its cancer-associated splice variant could be translated in vitro and in mammalian cells into stable proteins. Other results showed that selection by exosomes of the splice variant mRNA could be regulated by an external stimulus. Thus, a broad and diverse regulated pool of tRNA synthetase-derived mRNAs is packaged for genetic exchange.     

Effect of mutations affecting lysyl-tRNAlys on the regulation of lysine biosynthesis in Escherichia coli.

Summary When studying mutants affecting lysyl-tRNA synthetase or tRNA^Lys (hisT, hisW), a lack of correlation is clearly observed between the amount of lysyl-tRNA and the level of derepression of several lysine biosynthetic enzymes. This excludes the possible role of lysyl-tRNA as the specific corepressor of the lysine regulon. However, the level of derepression of DAP-decarboxylase, the last enzyme of the lysine pathway, is very low in the hisT mutant; this indicates that tRNA^Lys is a secondary effector involved in the regulation of the synthesis of this enzyme.Abbreviations DAP diaminopimelate - KRS lysyl-tRNA synthetase - L-lysine tRNA ligase (AMP) (EC6.1.16) - AK III lysinesensitive aspartokinase (EC 2.7.24) - ASA-dehydrogenase aspartic semialdehyde dehydrogenase (EC 1.2.1.10) - DHDP-reductase dihydrodipicolinic acid reductase - DAP-decarboxylase diaminopimelate decarboxylase (EC 4.1.1.20) - AK I threonine-sensitive aspartokinase - HDHI threonine-sensitive homoserine dehydrogenase     

S-adenosylhomocysteine hydrolases as the primary target enzymes in androgen regulation of methylation complexes

tRNA methylation complexes consisting of S-adenosylmethionine (AdoMet) synthetase, tRNA methylases, and S-adenosylhomocysteine (AdoHcy) hydrolase have been prepared from rat Novikoff hepatoma cells. The existence of the ternary enzyme complex is supported by dissociation and reconstitution of the ternany tRNA methylation complexes. In rat prostate and testis, two isozymes each for AdoMet synthetase and AdoHcy hydrolase are detected. The K_m (methionine) values for the two AdoMet synthetases are 3.1 and 23.7 μm and the K_m (adenosine) values for the two AdoHcy hydrolases are 0.33 and 1.8 μm. Correspondingly, two groups of methylation complexes are detectable, sedimenting in a sucrose gradient as 7 S and 8 S. The 7 S complexes are composed of AdoMet synthetase and AdoHcy hydrolase with the higher K_m values, and the 8 S complexes are composed of the respective isozymes with the lower K_m values. tRNA methylation complexes belong to the 8 S group. In hormone-depleted rat prostates and testes following hypophysectomy, the specific activities of AdoMet synthetases, tRNA methylases, and AdoHcy hydrolases are decreased severely, but are restored promptly after administration of testosterone. Thus, methylation enzymes are responsive to the regulation by steroid hormone. AdoHcy hydrolases from hormone-depleted tissues are unstable, and ternary tRNA methylation complexes are easily dissociable into individual activities. The stability of AdoHcy hydrolases is markedly improved by testosterone, and the integrity of ternary tRNA methylation complexes is maintained in the presence of testosterone. These results suggest that AdoHcy hydrolases are the primary target enzymes in adrogen regulation of methylation complexes.     

Active site of lysyl-tRNA synthetase: structural studies of the adenylation reaction

Aminoacyl-tRNA synthetases play a key role in protein biosynthesis by catalyzing the specific aminoacylation of tRNA. The energy required for the formation of the ester bond between the amino acid carboxylate group and the tRNA acceptor stem is supplied by coupling the reaction to the hydrolysis of ATP. Lysyl-tRNA synthetase from Escherichia coli belongs to the family of class II synthetases and carries out a two-step reaction, in which lysine is activated by being attached to the alpha-phosphate of AMP before being transferred to the cognate tRNA. Crystals of the thermo-inducible E. coli lysyl-tRNA synthetase LysU which diffract to 2.1 A resolution have been used to determine crystal structures of the enzyme in the presence of lysine, the lysyl-adenylate intermediate, and the nonhydrolyzable ATP analogue AMP-PCP. Additional data have been obtained from crystals soaked in a solution containing ATP and Mn(2+). The refined crystal structures give "snapshots" of the active site corresponding to key steps in the aminoacylation reaction and provide the structural framework for understanding the mechanism of lysine activation. The active site of LysU is shaped to position the substrates for the nucleophilic attack of the lysine carboxylate on the ATP alpha-phosphate. No residues are directly involved in catalysis, but a number of highly conserved amino acids and three metal ions coordinate the substrates and stabilize the pentavalent transition state. A loop close to the catalytic pocket, disordered in the lysine-bound structure, becomes ordered upon adenine binding.