A general suppressor of RNA polymerase I,II and III mutations in Saccharomyces cerevisiae

The pem locus, which is responsible for the stable maintenance of the low copy number plasmid R100, contains the pemK gene, whose product has been shown to be a growth inhibitor. Here, we attempted to isolate mutants which became tolerant to transient induction of the PemK protein. We obtained 20 mutants (here called pkt for PemK tolerance), of which 9 were temperature sensitive for growth. We analyzed the nine mutants genetically and found that they could be classified into three complementation groups, pktA, pktB and pktC, which corresponded to three genes, ileS, gltX and asnS, encoding isoleucyl-, glutamyl- and asparaginyl-tRNA synthetases, respectively. Since these aminoacyl-tRNA synthetase mutants did not produce the PemK protein upon induction at the restrictive temperature, these mutants could be isolated because they behaved as if they were tolerant to the PemK protein. The procedure is therefore useful for isolating temperature-sensitive mutants of aminoacyl-tRNA synthetases.     

Subcellular distribution and properties of rabbit liver aminoacyl-tRNA synthetases under myocardial ischemia

Subcellular distribution of aminoacyl-tRNA synthetase activities has been studied in normal rabbit liver and under experimental myocardial ischemia (EMI). An increase in the activity of a number of aminoacyl-tRNA synthetases in postmitochondrial and postribosomal supernatants from rabbit liver has been determined 12 hr after EMI. Gel chromatography of the postribosomal supernatant on Sepharose 6B shows that aminoacyl-tRNA synthetase activities are distributed among the fractions with M_r 1.82×10⁶, 0.84×10⁶ (high-M_r aminoacyl-tRNA synthetase complexes) and 0.12–0.35×10⁶. In the case of EMI aminoacyl-tRNA synthetase activities are partly redistributed from the 1.82×10⁶ complex into the 0.84×10⁶ complex. The catalytic properties of both free and complex leucyl-tRNA synthetases have been compared. K_M for all the substrates are the values of the same order in norm and under EMI. A decrease in some aminoacyl-tRNA synthetase activities associated with polyribosomes has been observed 12 hr after EMI. The interaction of aminoacyl-tRNA synthetases with polyribosomes stimulates the catalytic activity of some enzymes and protects them from heat inactivationin vitro. It is assumed that the changes in association of aminoacyl-tRNA synthetases with high-M_r complexes and compartmentalization of these enzymes on polyribosomes may be related to the alteration of protein biosynthesis under myocardial ischemia.     

Transfer RNA Identity Change in Anticodon Variants of E. coli tRNAPhe in Vivo

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA^Phe. Constructing different anticodon mutants of E. coli tRNA^Phe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA^Phe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA^Phe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA^Phe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.     

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.     

Sequence, structure and evolutionary relationships between class 2 aminoacyl-tRNA synthetases: An update

The seven class 2 aminoacyl-tRNA synthetases that are α₂ dimers have previously been divided by sequence homology into class 2a (seryl-, threonyl-, prolyl- and histidyl-) and class 2b (aspartyl-, asparaginyl- and lysyl-). It has been more difficult to classify the glycyl-, phenylalanyl- and alanyl-tRNA synthetases which have different subunit stoichiometries and which did not apparently contain all three canonical class 2 motifs. New sequence and structural information relating to the three problematic synthetases will be discussed permitting a step forward to be taken in the understanding of the evolutionary relationships between the class 2 synthetases.     

The stable maintenance system pem of plasmid R100: degradation of PemI protein may allow PemK protein to inhibit cell growth.

We constructed plasmids carrying heat-inducible pemI and pemK genes, which were fused with the collagen-lacZ sequence in frame. The PemK-collagen-LacZ (PemK*) protein produced from the fusion gene upon heat induction inhibited the growth of cells and killed most of the cells in the absence of the PemI protein but did not do so in the presence of the PemI protein. This supports our previous assumption that the PemK protein inhibits cell division, leading to cell death, whereas the PemI protein suppresses the function of the PemK protein. We also constructed the plasmid carrying the heat-inducible pem operon which consists of the intact pemI gene and the pemK gene fused with collagen-lacZ. The simultaneously induced PemI and PemK* proteins did not inhibit the growth of cells. However, the temperature shift to 30 degrees C after induction of both proteins at 42 degrees C caused inhibition of cell growth and death of most cells. This suggests that the PemI protein is somehow inactivated upon the arrest of de novo synthesis of the PemI and PemK* proteins, allowing the PemK* protein to function. We observed that the PemI-collagen-LacZ (PemI*) protein was degraded faster than the PemK* protein, perhaps by the action of a protease(s). In fact, the lon mutation, which caused no apparent degradation of the PemI* protein, did not allow the PemK* protein to function, supporting the suggestion described above. Instability of the PemI protein would explain why the cells which have lost the pem+ plasmid are preferentially killed.     

The twenty aminoacyl-tRNA synthetases from Escherichia coli. General separation procedure, and comparison of the influence of pH and divalent cations on their catalytic activities

A general separation procedure of the twenty E. coli aminoacyl-tRNA synthetases including either a 105 000 g centrifugation or a polyethyleneglycol-dextran two-phases partition fractionation, and chromatographies on DEAE-cellulose, phosphocellulose and hydroxyapatite is described. The specific activities of the synthetases have been determined after each chromatographic step and compared to their respective activities in the 105 000 g supernatant. Some aminoacyl-tRNA synthetases were obtained at 80 per cent purity.The presence of phenylmethylsulfonyl fluoride does not significantly modify either the elution patterns of the synthetases during the various chromatographic steps or their specific activities. Thus, contrarily to enzymes from various eukaryotic organisms no significant inactivation of the E. coli aminoacyl-tRNA synthetases occurs via proteolytic processes during the purification procedure.The effects of various factors: pH, magnesium, and other bivalent cations including spermidine, were tested on the aminoacylation and the [³²P] PP_i-ATP isotope-exchange reactions, and the optimal aminoacylation and isotope-exchange conditions determined for 18 of the 20 E. coli aminoacyl-tRNA synthetases.     

Chemically modified ATP derivatives for the study of aminoacyl-tRNA synthetases from baker's yeast: ATP analogs with fixed conformations or modified triphosphate chains in the aminoacylation reaction

The systematic investigation of substrate specificity of aminoacyl-tRNA synthetases from yeast is completed by tests of ATP analogs with fixed conformation about the glycosidic bond and with modifications in the triphosphate chain as substrate analogs in the aminoacylation reaction. Two analogs with fixed high anti (8,2′-O-cyclo-ATP, 8,2′-S-cyclo-ATP) and two with fixed anti (8,3′-O-cyclo-ATP, 8,3′-S-cyclo-ATP) conformation have been tested in the esterification reaction of phenylalanyl-, seryl-, lysyl-, valyl-, isoleucyl-, arginyl-, and tyrosyl-tRNA synthetases from baker's yeast. None of the compounds was a substrate, whereas 11 K_i values could be determined. 8,2′-S-cyclo-ATP, remarkably, is the only analog which inhibits all these synthetases. Each compound with a fixed anti conformation inhibits two enzymes. Among 11 analogs with modifications in the triphosphate chain, four were substrates for phenylalanyl-, three for seryl-, one for lysyl-, three for valyl-, one for isoleucyl-, and none for arginyl- and for tyrosyl-tRNA synthetases. Two compounds were inhibitors of different types for phenylalanyl-, two for seryl-, seven for lysyl-, six for valyl-, nine for isoleucyl-, seven for arginyl-, and two for tyrosyl-tRNA synthetases. Their K_m, V, and K_i values have been determined. In the general picture of substrate specificity the subunit enzymes can tolerate substitutions in position 2, 2′, at the α-phosphorus, at the β,γ-P-X-P bridge and at the γ-phosphorus atom. The single chain enzymes tolerate substitutions in position 7 and at the γ-phosphorus. All seven synthetases from yeast need an intact NH₂ group in position 6 and an oxygen atom in position 3′.     

Molecular determinants of the yeast Arc1p-aminoacyl-tRNA synthetase complex assembly

Eukaryotic aminoacyl-tRNA synthetases are usually organized into high-molecular-weight complexes, the structure and function of which are poorly understood. We have previously described a yeast complex containing two aminoacyl-tRNA synthetases, methionyl-tRNA synthetase and glutamyl-tRNA synthetase, and one noncatalytic protein, Arc1p, which can stimulate the catalytic efficiency of the two synthetases. To understand the complex assembly mechanism and its relevance to the function of its components, we have generated specific mutations in residues predicted by a recent structural model to be located at the interaction interfaces of the N-terminal domains of all three proteins. Recombinant wild-type or mutant forms of the proteins, as well as the isolated N-terminal domains of the two synthetases, were overexpressed in bacteria, purified and used for complex formation in vitro and for determination of binding affinities using surface plasmon resonance. Moreover, mutant proteins were expressed as PtA or green fluorescent protein fusion polypeptides in yeast strains lacking the endogenous proteins in order to monitor in vivo complex assembly and their subcellular localization. Our results show that the assembly of the Arc1p-synthetase complex is mediated exclusively by the N-terminal domains of the synthetases and that the two enzymes bind to largely independent sites on Arc1p. Analysis of single-amino-acid substitutions identified residues that are directly involved in the formation of the complex in yeast cells and suggested that complex assembly is mediated predominantly by van der Waals and hydrophobic interactions, rather than by electrostatic forces. Furthermore, mutations that abolish the interaction of methionyl-tRNA synthetase with Arc1p cause entry of the enzyme into the nucleus, proving that complex association regulates its subcellular distribution. The relevance of these findings to the evolution and function of the multienzyme complexes of eukaryotic aminoacyl-tRNA synthetases is discussed.     

Two birds with one stone: genes that encode products targeted to two or more compartments

Eukaryotic cells are divided into multiple membrane-bound compartments, all of which contain proteins. A large subset of these proteins perform functions that are required in more than one compartment. Although in most cases proteins carrying out the same function in different compartments are encoded by different genes, this is not always true. Numerous examples have now been found where a single gene encodes proteins (or RNAs) found in two (or more) cell organelles or membrane systems. Some particularly clear examples come from protein synthesis itself: plant cells contain three protein-synthesizing compartments, the cytosol, the mitochondrial matrix and the plastid stroma. All three compartments thus require tRNAs and aminoacyl-tRNA synthetases. Some mitochondrial tRNAs and their aminoacyl-tRNA synthetases are identical to their cytosolic counterparts and they are encoded by the same genes. Similarly, some mitochondrial and plastid aminoacyl-tRNA synthetases are encoded by the same nuclear genes. The various ways in which differentially targeted products can be generated from single genes is discussed.     

EFFECTS OF CHRONIC ETHANOL INGESTION ON BRAIN AMINOACYL-tRNA SYNTHETASES AND tRNA

The effects of chronic ethanol ingestion on the in vivo aminoacylation of brain transfer RNA (tRNA) were examined in C57BL/6J mice. A pronounced inhibition in the formation of [¹⁴C]leucy]-tRNA and [¹⁴C]phenylalanyl-tRNA was observed in the ethanol drinking mice. Properties of aminoacyl-tRNA synthetases and tRNA were examined following their separation and isolation on a DEAE-cellulose column. Synthesis of [¹⁴C]leucyl-tRNA was found to have a complete dependence on ATP and Mg²⁺. Incubations were carried out by cross-matching tRNA from control rat brain with synthetases obtained from the brains of control or ethanol-drinking mice. Under these conditions, a decreased ability for aminoacylation could be demonstrated when the source of enzyme was derived from ethanol-treated brain. The data indicate that the major effect of ethanol ingestion on the aminoacylation reaction is exerted on aminoacyl-tRNA synthetases.     

Roles of protein synthesis and tRNA aminoacylation in the regulation of intracellular protein breakdown in E. coli

The previously suggested roles of protein synthesis and tRNA aminoacylation in the regulation of intracellular protein breakdown were examined in strains of E. coli temperature-sensitive for aminoacyl-tRNA synthetases. Direct measurements of tRNA aminoacylation show no correlation between the degree of tRNA charging and the rate of protein breakdown. Protein breakdown was accelerated by transfer from 30°C to 42°C to about the same degree in temperature-sensitive mutants as in related normal strains. Deprivation of inorganic phosphate at the high temperature stimulated further protein breakdown in normal, but not in temperature-sensitive strains. It is concluded that the regulation of protein breakdown requires concomitant protein synthesis and is not influenced by the level of aminoacylation of tRNA.     

Identification of novel genes responsible for ethanol and/or thermotolerance by transposon mutagenesis in Saccharomyces cerevisiae

Saccharomyces cerevisiae strains tolerant to ethanol and heat stresses are important for industrial ethanol production. In this study, five strains (Tn 1–5) tolerant to up to 15% ethanol were isolated by screening a transposon-mediated mutant library. Two of them displayed tolerance to heat (42 °C). The determination of transposon insertion sites and Northern blot analysis identified seven putative genes (CMP2, IMD4, SSK2, PPG1, DLD3, PAM1, and MSN2) and revealed simultaneous down-regulations of CMP2 and IMD4, and SSK2 and PPG1, down-regulation of DLD3, and disruptions of the open reading frame of PAM1 and MSN2, indicating that ethanol and/or heat tolerance can be conferred. Knockout mutants of these seven individual genes were ethanol tolerant and three of them (SSK2, PPG1, and PAM1) were tolerant to heat. Such tolerant phenotypes reverted to sensitive phenotypes by the autologous or overexpression of each gene. Five transposon mutants showed higher ethanol production and grew faster than the control strain when cultured in rich media containing 30% glucose and initial 6% ethanol at 30 °C. Of those, two thermotolerant transposon mutants (Tn 2 and Tn 3) exhibited significantly enhanced growth and ethanol production compared to the control at 42 °C. The genes identified in this study may provide a basis for the application in developing industrial yeast strains.     

Inhibition of aminoacyl-tRNA synthetases by p-chloroamphetamine and its role in protein synthesis inhibition

p-Chloroamphetamine inhibited to some degree all amino acid-dependent pyrophosphate-exchange activities which could be detected in a rabbit reticulocyte extract. A detailed kinetic analysis of the reaction catalyzed by reticulocyte leucyl-tRNA synthetase demonstrated that the inhibitor affected only amino acid binding. Less rigorous studies of other synthetases from both rabbit reticulocyte and Escherichia coli could be similarly interpreted, suggesting that this compound interacts in a common manner with these several enzymes. The contribution of such effects to the inhibition of protein synthesis by the drug was investigated using cell-free translation systems in which rates of amino acid incorporation were limited to varying degrees by the synthesis and availability of aminoacyl-tRNA. In a wheat germ system programmed with globin mRNA, in which levels of amino acids and aminoacyl-tRNAs were shown to limit the rate of protein synthesis, the inhibition produced by p-chloroamphetamine could be partially reversed by increasing the concentration of the limiting amino acid. In a reticulocyte lysate, in which amino acid concentrations were not limiting, inhibition failed to show an amino acid-reversible component. Thus, while the inhibition of aminoacyl-tRNA synthetases by amphetamines can be shown in some cases to play a role in the effects of these compounds on in vitro protein synthesis, other sites of interference with initiation and/ or elongation reactions may predominate, depending on the construction of the system under study.     

氨酰tRNA 合成酶抑制剂作为新型抗感染药物的研究进展

细菌耐药性的不断上升对现有阶段的抗生素类药物提出了一个严峻的挑战,同时也掀起了针对于新靶标的抗菌药物的研究。氨酰tRNA合成酶(aaRS)催化特定氨基酸连接到相应的tRNA分子上,在蛋白质的合成过程中起着必不可少的作用。氨酰tRNA合成酶的抑制会导致蛋白质合成的停止,扰乱细菌和真菌的生长,因此氨酰tRNA合成酶是一类潜在的抗感染靶标。本文分别综述了天然产物及其衍生的aaRS抑制剂,底物和反应中间体模拟物,通过合成和通过虚拟筛选得到的aaRS抑制剂作为新型抗细菌和抗真菌药物的研究进展,并对aaRS的靶标特点、分类和催化机制作一简要介绍。     

Metabolic regulation of aminoacyl-tRNA synthetase biosynthesis in bakers' yeast.

The specific activities of 15 aminoacyl-tRNA synthetases in Saccharomyces cerevisiae were measured after growth under a variety of conditions that produced a range of cell-doubling times. The specific activity of each synthetase increased as cell-doubling time decreased. Control experiments eliminate the possibility that these results are due to preferential recovery of synthetases, or to the presence of activators in the faster growing cultures or inhibitors in the slower growing ones. These observations run counter to the expectation that synthetases in bacteria and yeast are negatively regulated by free amino acids, or, more likely, by aminoacyl-tRNA. In fact, as the growth medium was enriched, generation times decreased, and synthetase and aminoacyl-tRNA levels increased. It is suggested that cytoplasmic aminoacyl-tRNA synthetases may be more or less coordinately controlled such that their response to growth follows the pattern observed for ribosome production and RNA synthesis. This suggests the possibility of coordinated response of genes for components of the protein synthetic apparatus.     

Variations in activity of aminoacyl-tRNA synthetases as a function of development in Bacillus subtilis

Extracts from Bacillus sublilis cells at various stages of growth and spores were assayed for aminoacyl-tRNA synthetase and methionyl-tRNA transformylase activity. There was no major change in any synthetase activity or in methionyl-tRNA transformylase activity during the sporulation cycle, which implies that these are not sporulation induced enzymes. However, extracts from B. subtilis cultures showed a burst of activity of aminoacyl-tRNA synthetases during exponential growth.Preparations from dormant spores possessed the same kinds of aminoacyl-tRNA synthetase activities as vegetative cells for all the amino acids which were studied. Spores also contained methionyl-tRNA transformylases. These findings suggest that spores ought to be able to aminoacylate tRNA and formylate the initiator. N-formylmethionyl-tRNA, immediately upon germination.     

Horizontal Gene Transfer in Aminoacyl-tRNA Synthetases Including Leucine-Specific Subtypes

Aminoacyl-tRNA synthetases catalyze a fundamental reaction for the flow of genetic information from RNA to protein. Their presence in all organisms known today highlights their important role in the early evolution of life. We investigated the evolutionary history of aminoacyl-tRNA synthetases on the basis of sequence data from more than 200 Archaea, Bacteria, and Eukaryota. Phylogenetic profiles are in agreement with previous observations that many genes for aminoacyl-tRNA synthetases were transferred horizontally between species from all domains of life. We extended these findings by a detailed analysis of the history of leucyl-tRNA synthetases. Thereby, we identified a previously undetected case of horizontal gene transfer from Bacteria to Archaea based on phylogenetic profiles, trees, and networks. This means that, finally, the last subfamily of aminoacyl-tRNA synthetases has lost its exceptional position as the sole subfamily that is devoid of horizontal gene transfer. Furthermore, the leucyl-tRNA synthetase phylogenetic tree suggests a dichotomy of the archaeal/eukaryotic-cytosolic and bacterial/eukaryotic-mitochondrial proteins. We argue that the traditional division of life into Prokaryota (non-chimeric) and Eukaryota (chimeric) is favorable compared to Woese’s trichotomy into Archaea/Bacteria/Eukaryota. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Yves Van de Peer]     

On the Evolution of Structure in Aminoacyl-tRNA Synthetases

The aminoacyl-tRNA synthetases are one of the major protein components in the translation machinery. These essential proteins are found in all forms of life and are responsible for charging their cognate tRNAs with the correct amino acid. The evolution of the tRNA synthetases is of fundamental importance with respect to the nature of the biological cell and the transition from an RNA world to the modern world dominated by protein-enzymes. We present a structure-based phylogeny of the aminoacyl-tRNA synthetases. By using structural alignments of all of the aminoacyl-tRNA synthetases of known structure in combination with a new measure of structural homology, we have reconstructed the evolutionary history of these proteins. In order to derive unbiased statistics from the structural alignments, we introduce a multidimensional QR factorization which produces a nonredundant set of structures. Since protein structure is more highly conserved than protein sequence, this study has allowed us to glimpse the evolution of protein structure that predates the root of the universal phylogenetic tree. The extensive sequence-based phylogenetic analysis of the tRNA synthetases (Woese et al., Microbiol. Mol. Biol. Rev. 64:202-236, 2000) has further enabled us to reconstruct the complete evolutionary profile of these proteins and to make connections between major evolutionary events and the resulting changes in protein shape. We also discuss the effect of functional specificity on protein shape over the complex evolutionary course of the tRNA synthetases.     

Interaction of Colicins with Bacterial Cells III. Colicin-tolerant Mutations in Escherichia coli

Mutants that adsorb certain colicins without being killed, i.e., tolerant mutants (tol), were isolated from Escherichia coli K-12 strains. Selection was done either with colicin K or E2. Several groups of mutants showing different phenotypes were found, and some of them showed tolerance to both K and E colicins, which have different receptors. Many of these mutants mapped near gal. Typical mutants from group II, III, and IV were studied in more detail. The mutant loci were contransducible with gal by phage P1. The linkage order was deduced to be tol-gal-λ. In partially diploid strains, these mutant loci are recessive to wild-type alleles. Temperature-dependent conditionally tolerant mutants were also isolated. Two groups were found: the first was tolerant to E2 and E3 at 40 C, but sensitive at 30 C; the second was tolerant to E2 at 30 C, but sensitive at 40 C. Experiments done with these mutants suggest that these mutations affect the heat lability of some protein that is necessary for the response of cells to colicins. Conditionally lethal tolerant mutants were isolated which at 40 C were tolerant to E2 and E3 and could not grow, but which at 30 C were fully sensitive and grew normally. The mutation mapped near malA. The tolerance at 40 C is not due to a consequence of an inactivation of general cellular metabolism, but presumably is a cause of the subsequent inhibition of cellular growth. The results suggest that some protein components involved in the response to colicin are also vital to normal cellular growth.