How does Pseudomonas fluorescens avoid suicide from its antibiotic pseudomonic acid?: Evidence for two evolutionarily distinct isoleucyl-tRNA synthetases conferring self-defense

Two isoleucyl-tRNA synthetases (IleRSs) encoded by two distinct genes (ileS1 and ileS2) were identified in pseudomonic acid (mupirocin)-producing Pseudomonas fluorescens. The most striking difference between the two IleRSs (IleRS-R1 and IleRS-R2) is the difference in their abilities to resist pseudomonic acid. Purified IleRS-R2 showed no sensitivity to pseudomonic acid even at a concentration of 5 mm, 105 times higher than the Ki value of IleRS-R1. The amino acid sequence of IleRS-R2 exhibits eukaryotic features that are originally found in eukaryotic proteins. Escherichia coli cells transformed with the ileS2 gene exerted pseudomonic acid resistance more than did those transformed with ileS1. Cells transformed with both genes became almost as resistant as P. fluorescens. These results suggest that the presence of IleRS-R2 could be the major reason why P. fluorescens is intrinsically resistant to the antibiotic. Here we suggest that the evolutionary scenario of the eukaryotic ileS2 gene can be explained by gene acquisition and that the pseudomonic acid producer may have maintained the ileS2 gene to protect itself from pseudomonic acid.     

Modulation of substrate specificity within the amino acid editing site of leucyl-tRNA synthetase

The aminoacyl-tRNA synthetases covalently link transfer RNAs to their cognate amino acids. Some of the tRNA synthetases have evolved editing mechanisms to ensure fidelity in this first step of protein synthesis. The amino acid editing site for leucyl- (LeuRS) and isoleucyl- (IleRS) tRNA synthetases reside within homologous CP1 domains. In each case, a threonine-rich peptide and a second conserved GTG region that are separated by about 100 amino acids comprise parts of the hydrolytic editing site. While a number of sites are conserved between these two enzymes and likely confer a commonality to the mechanisms, some positions are idiosyncratic to LeuRS or IleRS. Herein, we provide evidence that a conserved arginine and threonine at respective sites in LeuRS and IleRS diverged to confer amino acid substrate recognition. This site complements other sites in the amino acid binding pocket of the editing active site of Escherichia coli LeuRS, including Thr252 and Val338, which collectively fine-tune amino acid specificity to confer fidelity.     

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.     

Plasticity of recognition of the 3'-end of mischarged tRNA by class I aminoacyl-tRNA synthetases

Certain aminoacyl-tRNA synthetases prevent potential errors in protein synthesis through deacylation of mischarged tRNAs. For example, the close homologs isoleucyl-tRNA synthetase (IleRS) and valyl-tRNA synthetase (ValRS) deacylate Val-tRNA(Ile) and Thr-tRNA(Val), respectively. Here we examined the chemical requirements at the 3'-end of the tRNA for these hydrolysis reactions. Single atom substitutions at the 2'- and 3'-hydroxyls of a variety of mischarged RNAs revealed that, while acylation is at the 2'-OH for both enzymes, IleRS catalyzes deacylation specifically from the 3'-OH and not from the 2'-OH. In contrast, ValRS can deacylate non-cognate amino acids from the 2'-OH. Moreover, for IleRS the specificity for a 3'-O location of the scissile ester bond could be forced to the 2'-position by introduction of a 3'-O-methyl moiety. Cumulatively, these and other results suggest that the editing sites of these class I aminoacyl-tRNA synthetases have a degree of inherent plasticity for substrate recognition. The ability to adapt to subtle differences in mischarged RNAs may be important for the high accuracy of aminoacylation.     

Basic faced alpha-helices are widespread in the peptide extensions of the eukaryotic aminoacyl-tRNA synthetases

Three aminoacyl-tRNA synthetases from yeast, one from plants and one from mammals possess unusual structures at their N termini, namely alpha helices with basic residues distributed asymmetrically, on a single face of the helix. It is unknown if these 'basic faced' alpha helices (BFAHs) are unique to the aminoacyl-tRNA synthetases. Analysis of the amino acid sequences of these five aminoacyl-tRNA synthetases using the hydrophobic moment algorithm failed to accurately identify the BFAHs. A new algorithm was therefore developed, called the 'basic moment'. This is a Fourier analysis procedure that predicts the distribution of basic residues within protein secondary structure. The basic moment identifies with a high degree of accuracy the five known BFAHs and also identifies further potential BFAHs at evolutionarily conserved positions in the peptide extensions of aspartyl-, lysyl- and valyl- tRNA synthetases from a range of eukaryotic species. In addition, the algorithm identifies the two-helix pair tRNA binding domain of alanyl-tRNA synthetase, implying that the domain includes a BFAH. The functional and evolutionary aspects of these structural features are discussed.     

Archaeabacterial Seryl-tRNA Synthetases: Adaptation to Extreme Environments and Evolutionary Analysis

The aminoacyl-tRNA synthetases are ubiquitous enzymes which catalyze a crucial step of the cell life, the specific attachment of amino acids to their cognate tRNA. The amino acid sequences of three archaeal seryl-tRNA synthetases (SerRS) from Haloarcula marismortui and Methanococcus jannaschii, both belonging to the group of Euryarchaeota, and from Sulfolobus solfataricus, of the group of Crenarchaeota, were aligned with other eubacterial and eukaryal available SerRS sequences. In an attempt to identify some features of adaptation to extreme environments of these organisms, amino acid composition and amino acid substitutions between mesophilic and thermophilic SerRS were analyzed. In addition, universal phylogenetic trees of SerRS including the three known archaeal sequences, rooted by the threonyl-tRNA synthetases were inferred. Amino acid analyses of the SerRS revealed two ways of adaptation to thermophilic environments between the Eubacteria and the Archaea; most of the usually described amino acid substitutions were nonsignificant in the case of archaeal thermophilic SerRS and most amino acid composition biases seemed to be linked to the genome G+C content pressure. The phylogenetic analysis of the SerRS showed the Archaea to be paraphyletic, H. marismortui emerging with the Gram-positive Bacteria, M. jannaschii being near the root of the tree, and S. solfataricus branching with Eucarya. Received: 30 March 1998 / Accepted: 14 July 1998     

Transiently misacylated tRNA is a primer for editing of misactivated adenylates by class I aminoacyl-tRNA synthetases

The genetic code depends on amino acid fine structure discrimination by aminoacyl-tRNA synthetases. For isoleucyl- (IleRS) and valyl-tRNA synthetases (ValRS), reactions that hydrolyze misactivated noncognate amino acids help to achieve high accuracy in aminoacylation. Two editing pathways contribute to aminoacylation fidelity: pretransfer and post-transfer. In pretransfer editing, the misactivated amino acid is hydrolyzed as an aminoacyl adenylate, while in post-transfer editing a misacylated tRNA is deacylated. Both reactions are dependent on a tRNA cofactor and require translocation to a site located approximately 30 A from the site of amino acid activation. Using a series of 3'-end modified tRNAs that are deficient in either aminoacylation, deacylation, or both, total editing (the sum of pre- and post-transfer editing) was shown to require both aminoacylation and deacylation activities. These and additional results with IleRS are consistent with a post-transfer deacylation event initiating formation of an editing-active enzyme/tRNA complex. In this state, the primed complex processively edits misactivated valyl-adenylate via the pretransfer route. Thus, misacylated tRNA is an obligatory intermediate for editing by either pathway.     

NMR analyses of the conformations of L-isoleucine and L-valine bound to Escherichia coli isoleucyl-tRNA synthetase

The 400-MHz 1H NMR spectra of L-isoleucine and L-valine were measured in the presence of Escherichia coli isoleucyl-tRNA synthetase (IleRS). Because of chemical exchange of L-isoleucine or L-valine between the free state and the IleRS-bound state, a transferred nuclear Overhauser effect (TRNOE) was observed among proton resonances of L-isoleucine or L-valine. However, in the presence of isoleucyl adenylate tightly bound to the amino acid activation site of IleRS, no TRNOE for L-isoleucine or L-valine was observed. This indicates that the observed TRNOE is due to the interaction of L-isoleucine or L-valine with the amino acid activation site of IleRS. The conformations of these amino acids in the amino acid activation site of IleRS were determined by the analyses of time dependences of TRNOEs and TRNOE action spectra. The IleRS-bound L-isoleucine takes the gauche+ form about the C alpha-C beta bond and the trans form about the C beta-C gamma 1 bond. The IleRS-bound L-valine takes the gauche- form about the C alpha-C beta bond. Thus, the conformation of IleRS-bound L-valine is the same as that of IleRS-bound L-isoleucine except for the delta-methyl group. The side chain of L-isoleucine or L-valine lies in an aliphatic hydrophobic pocket of the active site of IleRS. Such hydrophobic interaction with IleRS is more significant for L-isoleucine than for L-valine. The TRNOE analysis is useful for studying the amino acid discrimination mechanism of aminoacyl-tRNA synthetases.     

Ribosomal proteins in halobacteria

The amino acid sequences of 16 ribosomal proteins from archaebacterium Halobacterium marismortui have been determined by a direct protein chemical method. In addition, amino acid sequences of three proteins, S11, S18, and L25, have been established by DNA sequencing of their genes as well as by protein sequencing. Comparison of their sequences with those of ribosomal proteins from other organisms revealed that proteins S14, S16, S19, and L25 are related to both eukaryotic and eubacterial ribosomal proteins, being more homologous to eukaryotic than eubacterial counterparts, and proteins S12, S15, and L16 are related to only eukaryotic ribosomal proteins. Furthermore, some proteins are found to be similar to only eubacterial proteins, whereas other proteins show no homology to any other known ribosomal proteins. Comparisons of amino acid compositions between halophilic and nonhalophilic ribosomal proteins revealed that halophilic proteins gain aspartic and glutamic acid residues and significantly lose lysine and arginine residues. In addition, halophilic proteins seem to lose isoleucine as compared with Escherichia coli ribosomal proteins.     

Partitioning of tRNA-dependent Editing between Pre- and Post-transfer Pathways in Class I Aminoacyl-tRNA Synthetases

Hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes occurs in a spatially separate domain inserted into the catalytic Rossmann fold, but the location and mechanisms of pre-transfer hydrolysis of misactivated amino acids have been uncertain. Here, we use novel kinetic approaches to distinguish among three models for pre-transfer editing by Escherichia coli isoleucyl-tRNA synthetase (IleRS). We demonstrate that tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme and that noncatalytic hydrolysis after release is too slow to account for the observed rate of clearing. Measurements of the microscopic rate constants for amino acid transfer to tRNA in IleRS and the related valyl-tRNA synthetase (ValRS) further suggest that pre-transfer editing in IleRS is an enzyme-catalyzed activity residing in the synthetic active site. In this model, the balance between pre-transfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate. Rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS, whereas in ValRS transfer to tRNA is 200-fold faster than hydrolysis. In consequence, editing by ValRS occurs nearly exclusively by post-transfer hydrolysis in the editing domain, whereas in IleRS both pre- and post-transfer editing are important. In both enzymes, the rates of amino acid transfer to tRNA are similar for cognate and noncognate aminoacyl-adenylates, providing a significant contrast with editing DNA polymerases.     

Negative catalysis by the editing domain of class I aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (AARS) translate the genetic code by loading tRNAs with the cognate amino acids. The errors in amino acid recognition are cleared at the AARS editing domain through hydrolysis of misaminoacyl-tRNAs. This ensures faithful protein synthesis and cellular fitness. Using Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme, we demonstrated that the class I editing domain clears the non-cognate amino acids well-discriminated at the synthetic site with the same rates as the weakly-discriminated fidelity threats. This unveiled low selectivity suggests that evolutionary pressure to optimize the rates against the amino acids that jeopardize translational fidelity did not shape the editing site. Instead, we propose that editing was shaped to safeguard cognate aminoacyl-tRNAs against hydrolysis. Misediting is prevented by the residues that promote negative catalysis through destabilisation of the transition state comprising cognate amino acid. Such powerful design allows broad substrate acceptance of the editing domain along with its exquisite specificity in the cognate aminoacyl-tRNA rejection. Editing proceeds by direct substrate delivery to the editing domain (in cis pathway). However, we found that class I IleRS also releases misaminoacyl-tRNA^Ile and edits it in trans. This minor editing pathway was up to now recognized only for class II AARSs.     

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: Phylogenetic implications

Summary The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%–49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.     

Inhibition of aminoacyl-tRNA synthetases by the mycotoxin patulin

The effect of patulin on tRNA aminoacylation has been determined. This mycotoxin inhibits the aminoacylation process by irreversibly inactivating aminoacyl-tRNA synthetases. At neutral and alkaline pH-values, the inactivation occurs mainly by modification of essential thiol groups of the protein, whereas at acidic pH, where the effect is the most pronounced, the modification of other amino acid residues cannot be excluded.     

A component of the multisynthetase complex is a multifunctional aminoacyl-tRNA synthetase.

In higher eukaryotes, nine aminoacyl-tRNA synthetases are associated within a multienzyme complex which is composed of 11 polypeptides with molecular masses ranging from 18 to 150 kDa. We have cloned and sequenced a cDNA from Drosophila encoding the largest polypeptide of this complex. We demonstrate here that the corresponding protein is a multifunctional aminoacyl-tRNA synthetase. It is composed of three major domains, two of them specifying distinct synthetase activities. The amino and carboxy-terminal domains were expressed separately in Escherichia coli, and were found to catalyse the aminoacylation of glutamic acid and proline tRNA species, respectively. The central domain is made of six 46 amino acid repeats. In prokaryotes, these two aminoacyl-tRNA synthetases are encoded by distinct genes. The emergence of a multifunctional synthetase by a gene fusion event seems to be a specific, but general attribute of all higher eukaryotic cells. This type of structural organization, in relation to the occurrence of multisynthetase complexes, could be a mechanism to integrate several catalytic domains within the same particle. The involvement of the internal repeats in mediating complex assembly is discussed.     

A present-day aminoacyl-tRNA synthetase with ancestral editing properties

Leucyl-, isoleucyl-, and valyl-tRNA synthetases form a subgroup of related aminoacyl-tRNA synthetases that attach similar amino acids to their cognate tRNAs. To prevent amino acid misincorporation during translation, these enzymes also hydrolyze mischarged tRNAs through a post-transfer editing mechanism. Here we show that LeuRS from the deep-branching bacterium Aquifex aeolicus edits the complete set of aminoacylated tRNAs generated by the three enzymes: Ile-tRNA(Ile), Val-tRNA(Ile), Val-tRNA(Val), Thr-tRNA(Val), and Ile-tRNA(Leu). This unusual enlarged editing property was studied in a model of a primitive editing system containing a composite minihelix carrying the triple leucine, isoleucine, and valine identity mimicking the primitive tRNA precursor. We found that the freestanding LeuRS editing domain can edit this precursor in contrast to IleRS and ValRS editing domains. These results suggest that A. aeolicus LeuRS carries editing properties that seem more primitive than those of IleRS and ValRS. They suggest that the A. aeolicus editing domain has preserved the ambiguous editing property from the ancestral common editing domain or, alternatively, that this plasticity results from a specific metabolic adaptation.     

Dual targeting of aminoacyl-tRNA synthetases to the apicoplast and cytosol in Plasmodium falciparum

The causative agent of malaria, Plasmodium, possesses three translationally active compartments: the cytosol, the mitochondrion and a relic plastid called the apicoplast. Aminoacyl-tRNA synthetases to charge tRNA are thus required for all three compartments. However, the Plasmodiumfalciparum genome encodes too few tRNA synthetases to supply a unique enzyme for each amino acid in all three compartments. We have investigated the subcellular localisation of three tRNA synthetases (AlaRS, GlyRS and ThrRS), which occur only once in the nuclear genome, and we show that each of these enzymes is dually localised to the P. falciparum cytosol and the apicoplast. No mitochondrial fraction is apparent for these three enzymes, which suggests that the Plasmodium mitochondrion lacks at least these three tRNA synthetases. The unique Plasmodium ThrRS is the presumed target of the antimalarial compound borrelidin. Borrelidin kills P. falciparum parasites quickly without the delayed death effect typical of apicoplast translation inhibitors and without an observable effect on apicoplast morphology. By contrast, mupirocin, an inhibitor of the apicoplast IleRS, kills with a delayed death effect that inhibits apicoplast growth and division. Because inhibition of dual targeted tRNA synthetases should arrest translation in all compartments of the parasite, these enzymes deserve further investigation as potential targets for antimalarial drug development.     

The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

Here we describe the many applications of acid urea polyacrylamide gel electrophoresis (acid urea PAGE) followed by Northern blot analysis to studies of tRNAs and aminoacyl-tRNA synthetases. Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Among the examples described are (i) analysis of the effect of mutations in the Escherichia coli initiator tRNA on its aminoacylation and formylation; (ii) evidence of orthogonality of suppressor tRNAs in mammalian cells and yeast; (iii) analysis of aminoacylation specificity of an archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNA(Pro) with cysteine, but does not aminoacylate archaeal tRNA(Cys) with cysteine; (iv) identification and characterization of the AUA-decoding minor tRNA(Ile) in archaea; and (v) evidence that the archaeal minor tRNA(Ile) contains a modified base in the wobble position different from lysidine found in the corresponding eubacterial tRNA.     

Active site of an aminoacyl-tRNA synthetase dissected by energy-transfer-dependent fluorescence.

Aminoacyl-tRNA synthetases establish the rules of the genetic code by catalyzing attachment of amino acids to specific transfer RNAs (tRNAs) that bear the anticodon triplets of the code. Each of the 20 amino acids has its own distinct aminoacyl-tRNA synthetase. Here we use energy-transfer-dependent fluorescence from the nucleotide probe N-methylanthraniloyl dATP (mdATP) to investigate the active site of a specific aminoacyl-tRNA synthetase. Interaction of the enzyme with the cognate amino acid and formation of the aminoacyl adenylate intermediate were detected. In addition to providing a convenient tool to characterize enzymatic parameters, the probe allowed investigation of the role of conserved residues within the active site. Specifically, a residue that is critical for binding could be distinguished from one that is important for the transition state of adenylate formation. Amino acid binding and adenylate synthesis by two other aminoacyl-tRNA synthetases was also investigated with mdATP. Thus, a key step in the synthesis of aminoacyl-tRNA can in general be dissected with this probe.     

Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo.

The development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the synthesis of proteins with novel structures and activities. Our approach to this problem consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are not catalytically competent with all the endogenous Escherichia coli tRNAs and aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. To evolve the desired amino acid specificity, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers high specificity for the amino acid of interest, eliminating the potential for contamination with synthetases active towards wild-type amino acids in multiple rounds of selection.     

Primary structures of ribosomal proteins from the archaebacterium Halobacterium marismortui and the eubacterium Bacillus stearothermophilus.

Approximately 40 ribosomal proteins from each Halobacterium marismortui and Bacillus stearothermophilus have been sequenced either by direct protein sequence analysis or by DNA sequence analysis of the appropriate genes. The comparison of the amino acid sequences from the archaebacterium H marismortui with the available ribosomal proteins from the eubacterial and eukaryotic kingdoms revealed four different groups of proteins: 24 proteins are related to both eubacterial as well as eukaryotic proteins. Eleven proteins are exclusively related to eukaryotic counterparts. For three proteins only eubacterial relatives-and for another three proteins no counterpart-could be found. The similarities of the halobacterial ribosomal proteins are in general somewhat higher to their eukaryotic than to their eubacterial counterparts. The comparison of B stearothermophilus proteins with their E coli homologues showed that the proteins evolved at different rates. Some proteins are highly conserved with 64-76% identity, others are poorly conserved with only 25-34% identical amino acid residues.