Mitochondrial leucine tRNA level and PTCD1 are regulated in response to leucine starvation

Pentatricopeptide repeat domain protein 1 (PTCD1) is a novel human protein that was recently shown to decrease the levels of mitochondrial leucine tRNAs. The physiological role of this regulation, however, remains unclear. Here we show that amino acid starvation by leucine deprivation significantly increased the mRNA steady-state levels of PTCD1 in human hepatocarcinoma (HepG2) cells. Amino acid starvation also increased the mitochondrially encoded leucine tRNA (tRNA^Leu(CUN)) and the mRNA for the mitochondrial leucyl-tRNA synthetase (LARS2). Despite increased PTCD1 mRNA steady-state levels, amino acid starvation decreased PTCD1 on the protein level. Decreasing PTCD1 protein concentration increases the stability of the mitochondrial leucine tRNAs, tRNA^Leu(CUN) and tRNA^Leu(UUR) as could be shown by RNAi experiments against PTCD1. Therefore, it is likely that decreased PTCD1 protein contributes to the increased tRNA^Leu(CUN) levels in amino acid-starved cells. The stabilisation of the mitochondrial leucine tRNAs and the upregulation of the mitochondrial leucyl-tRNA synthetase LARS2 might play a role in adaptation of mitochondria to amino acid starvation.     

Similarities in properties and a functional difference in purified leucyl-tRNA synthetase isolated from two developmental stages of Tenebrio molitor

In Tenebrio molitor, as well as in other biological systems, there are indications that differences in leucyl-tRNA synthetase activity may play a role in translational control. However, it has not been clear whether the difference in activity is due to the appearance of a multiplicity of enzymes during development or to the alteration of a single enzyme.The purification of leucyl-tRNA synthetase from day 1 and day 7 after the larval pupal molt of Tenebrio molitor is described. The enzyme from both developmental stages was purified over a 1000-fold. The two enzyme preparations are identical in molecular weight (99,000). They show the same characteristics after aging. The pH optimum, heat inactivation behavior, and dependency on divalent cations are the same for both enzymes. They also show identical kinetics with similar values of Km for leucine, ATP, Mg²⁺, and tRNA day 1. However, leucyl-tRNA synthetase purified from day 7 exhibits an additional function in recognizing a new species of isoaccepting tRNA in day 7 tRNA. We have tentatively concluded that the two enzymes are probably different forms of the same enzyme and the additional activity is due to alteration of the enzyme at the macromolecular level during development.     

Derepression of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

The kinetics of derepression of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined during valine-, isoleucine-, and leucine-limited growth. When valine was limiting growth, valyl-tRNA synthetase formation was maximally derepressed within 5 min, whereas the rates of synthesis of isoleucyl-, and leucyl-tRNA synthetases were unchanged. Isoleucine-restricted growth caused a maximal derepression of isoleucyl-tRNA synthetase formation in 5 min and derepression of valyl-tRNA synthetase formation in 15 min with no effect on leucyl-tRNA synthetase formation. When leucine was limiting growth, leucyl-tRNA synthetase formation was immediately derepressed, whereas valyl- and isoleucyl-tRNA synthetase formation was unaffected by manipulation of the leucine supply to the cells. These results support our previous findings that valyl-tRNA synthetase formation is subject to multivalent repression control by both isoleucine and valine. In contrast, repression control of iso-leucyl- and leucyl-tRNA synthetase formation is specifically mediated by the supply of the cognate amino acid.     

Specific degradation of a plant leucyl transfer ribonucleic Acid by a factor in the homologous synthetase preparation

Partially purified aminoacyl synthetase preparations from pea roots (Pisum sativum L. var. Alaska) contain a heat-labile factor which can degrade leucyl-tRNA₆^leu to a new species. The singular electrophoretic and chromatographic mobilities, the isoprenoid nucleoside content, and the charging characteristics of the new species (designated leucyl-tRNA_L^leu), suggest that it is a fragment of tRNA₆^leu containing at least that portion of the original molecule extending from the 3′ terminus to the anticodon. Conversion appears to be highly specific since neither bulk tRNA, the other leucine tRNA subspecies, nor tyrosine, phenylalanine, or tryptophan tRNAs are susceptible to degradation during incubation with the synthetase preparation.     

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.     

Patterns of E. coli leucine tRNA isoacceptors following bacteriophage MS2 infection.

The RNA extracted from MS2 phage particles can accept radioactive leucine and serine in the presence of tRNA activating enzymes. Leucine acceptance is due to the presence of E. coli leucine tRNA that binds very tightly to the virus particle. RPC-5 column chromatography shows that the pattern of virus associated leucyl-tRNA isoacceptors is different from that of normal E. coli leucyl-tRNA. It is also different from the pattern of host leucyl-tRNA isoacceptors found in E. coli lysate following MS2 phage infection. The RPC-5 pattern of the latter tRNA shows several new peaks of leucine tRNA isoacceptors. The possibility that these tRNAs are some modified forms of normal leucine tRNA isoacceptors is suggested.     

Isolation and binding properties of leucyl-tRNA synthetase from Escherichia coli MRE 600

Summary A procedure for the large-scale isolation of leucyl-tRNA synthetase from E. coli MRE 600 is described: The enzyme was purified about 320-fold to homogeneity by precipitation with cetyl-trimethyl-ammonium bromide, two consecutive chromatographies on DEAE-cellulose and three on hydroxyapatite with an over-all yield of 4%.The molecular weight of leucyl-tRNA synthetase from E. coli MRE 600 was found to be 99 000 daltons. Binding studies by ultracentrifugation and equilibrium partition showed that the enzyme binds leucine, leucyl-adenylate and tRNA^Leu, each in a 1 : 1 stoichiometry. For ATP only a very weak binding to the enzyme could be observed, which did not allow the evaluation of the complex stoichiometry. The presence of ATP was not required for the binding of leucine or tRNA to leucyl-tRNA synthetase from E. coli MRE 600.     

Evidence That the Majority of Leucine Transfer Ribonucleic Acid Is Not Involved in Repression in Salmonella typhimurium

Leucine transfer ribonucleic acid (tRNA) was almost fully charged, and the isoleucine-valine and leucine enzymes remained derepressed when trifluoroleucine was added to a leucine auxotroph. High levels of charged leucine tRNA and derepression were also found in a leucyl-tRNA synthetase mutant.     

Ribosomal frameshifting in the yeast retrotransposon Ty: tRNAs induce slippage on a 7 nucleotide minimal site

Ribosomal frameshifting regulates expression of the TYB gene of yeast Ty retrotransposons. We previously demonstrated that a 14 nucleotide sequence conserved between two families of Ty elements was necessary and sufficient to support ribosomal frameshifting. This work demonstrates that only 7 of these 14 nucleotides are needed for normal levels of frameshifting. Any change to the sequence CUU-AGG-C drastically reduces frameshifting; this suggests that two specific tRNAs, tRNA(UAGLeu) and tRNA(CCUArg), are involved in the event. Our tRNA overproduction data suggest that a leucyl-tRNA, probably tRNA(UAGLeu), an unusual leucine isoacceptor that recognizes all six leucine codons, slips from CUU-Leu onto UUA-Leu (in the +1 reading frame) during a translational pause at the AGG-Arg codon induced by the low availability of tRNA(CCUArg), encoded by a single-copy essential gene. Frameshifting is also directional and reading frame specific. Interestingly, frameshifting is inhibited when the "slip" CUU codon is located three codons downstream, but not four or more codons downstream, of the translational initiation codon.     

Solution conformation of several free tRNALeu species from bean, yeast and Escherichia coli and interaction of these tRNAs with bean cytoplasmic Leucyl-tRNA synthetase. A phosphate alkylation study with ethylnitrosourea.

The solution conformation of eight leucine tRNAs from Phaseolus vulgaris, baker's yeast and Escherichia coli, characterized by long variable regions, and the interaction of four of them with bean cytoplasmic leucyl-tRNA synthetase were studied by phosphate mapping with ethylnitrosourea. Phosphate reactivities in the variable regions agree with the existence of RNA helices closed by miniloops. At the junction of these regions with the T-stem, phosphate 48 is strongly protected, in contrast to small variable region tRNAs where P49 is protected. The constant protection of P22 is another characteristics of leucine tRNAs. Conformational differences between leucine isoacceptors concern the anticodon region, the D-arm and the variable region. In several parts of free tRNALeu species, e.g. in the T-loop, phosphate reactivities are similar to those found in tRNAs of other specificities, indicating conformational similarities among tRNAs. Phosphate alkylation of four leucine tRNAs complexed to leucyl-tRNA synthetase indicates that the 3'-side of the anticodon stem, the D-stem and the hinge region between the anticodon and D-stems are in contact with the plant enzyme.     

Leucyl-transfer ribonucleic acid synthetase from a wild-type and temperature-sensitive mutant of Salmonella typhimurium

Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following K(m) values: leucine, 1.1 x 10(-5)m; adenosine triphosphate, 6.5 x 10(-4)m; tRNA(I) (Leu), 4.1 x 10(-8)m; tRNA(II) (Leu), 4.3 x 10(-8)m; tRNA(III) (Leu), 5.3 x 10(-8)m; and tRNA(IV) (Leu), 2.9 x 10(-8)m. The tRNA(Leu) fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min(-1). Strain CV356 (leuS3 leuABCD702 ara-9 gal-205) was isolated as a mutant resistant to dl-4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium.     

Isolation and characterization of revertants of the mammalian temperature sensitive leucyl-tRNA synthetase mutant tsHl.

Nine spontaneous and seven ethyl methanesulfonate induced revertants of the Chinese hamster ovary cell line mutant (tsHl), which possesses a temperature sensitive leucyl-tRNA synthetase, were isolated and characterized with respect to growth rate, leucyl-tRNA synthetase activity and thermolability, intracellular leucine pool size, and rRNA content. Although most revertants had increased leucyl-tRNA synthetase activity, and of those tested, all but one had increased thermostability, each appears to be unique. One revertant may be an intergenic suppressor since it appears to contain an elevated level of tsHl-like synthetase. There was no evidence for any of the revertants having increased rRNA and tRNA contents, however, many showed leucine pools two to three times larger than wild type cells. Since similar increases have been observed in tsHl cells they are believed to result from regulation of leucine pool size by the leucyl-tRNA synthetase and are of a magnitude sufficient to affect significantly the growth of revertants at 38.5 degrees C.     

Inhibition of leucyl-tRNA synthetase in Escherichia coli by the cytostatic 5,8-dioxo-6-amino-7-chloroquinoline.

At concentrations of 1-1.6 mug/ml, 5,8-dioxo-6-amino-7-chloroquinoline causes auxotrophy for leucine in Escherichia coli MRE 600. With increasing concentrations of this quinone additional amino acids are required for growth. The amount of leucine in the pool of free amino acids is not decreased after treatment of E. coli with the quinone. Transfer RNALeu, however, is charged with leucine less than 10% in quinone-treated cells of E. coli, whereas in control cells the degree of aminoacylation is about 85%. From these data we conclude that the quinone causes auxotrophy for leucine by interacting with the charging process of tRNALeu. Quinone was found to inhibit leucyl-tRNA synthetase activity in purified extracts of E. coli with E. coli tRNA as substrate.     

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].     

The Yin and Yang of tRNA: proper binding of acceptor end determines the catalytic balance of editing and aminoacylation

Faithful translation of the genetic code depends on accurate coupling of amino acids with cognate transfer RNAs (tRNAs) catalyzed by aminoacyl-tRNA synthetases. The fidelity of leucyl-tRNA synthetase (LeuRS) depends mainly on proofreading at the pre- and post-transfer levels. During the catalytic cycle, the tRNA CCA-tail shuttles between the synthetic and editing domains to accomplish the aminoacylation and editing reactions. Previously, we showed that the Y330D mutation of Escherichia coli LeuRS, which blocks the entry of the tRNA CCA-tail into the connective polypeptide 1domain, abolishes both tRNA-dependent pre- and post-transfer editing. In this study, we identified the counterpart substitutions, which constrain the tRNA acceptor stem binding within the synthetic active site. These mutations negatively impact the tRNA charging activity while retaining the capacity to activate the amino acid. Interestingly, the mutated LeuRSs exhibit increased global editing activity in the presence of a non-cognate amino acid. We used a reaction mimicking post-transfer editing to show that these mutations decrease post-transfer editing owing to reduced tRNA aminoacylation activity. This implied that the increased editing activity originates from tRNA-dependent pre-transfer editing. These results, together with our previous work, provide a comprehensive assessment of how intra-molecular translocation of the tRNA CCA-tail balances the aminoacylation and editing activities of LeuRS.     

Transfer RNA: A dancer between charging and mis-charging for protein biosynthesis

Transfer RNA plays a fundamental role in the protein biosynthesis as an adaptor molecule by functioning as a biological link between the genetic nucleotide sequence in the mRNA and the amino acid sequence in the protein. To perform its role in protein biosynthesis, it has to be accurately recognized by aminoacyl-tRNA synthetases (aaRSs) to generate aminoacyl-tRNAs (aa-tRNAs). The correct pairing between an amino acid with its cognate tRNA is crucial for translational quality control. Production and utilization of mis-charged tRNAs are usually detrimental for all the species, resulting in cellular dysfunctions. Correct aa-tRNAs formation is collectively controlled by aaRSs with distinct mechanisms and/or other trans-factors. However, in very limited instances, mis-charged tRNAs are intermediate for specific pathways or essential components for the translational machinery. Here, from the point of accuracy in tRNA charging, we review our understanding about the mechanism ensuring correct aa-tRNA generation. In addition, some unique mis-charged tRNA species necessary for the organism are also briefly described.     

Pyrophosphate-caused inhibition of the aminoacylation of tRNA by the leucyl-tRNA synthetase from Neurospora crassa

Inorganic pyrophosphate inhibits the aminoacylation of tRNALeu by the leucyl-tRNA synthetase from Neurospora crassa giving very low Kapp.i, PPi values of 3-20 microM. The inhibition by pyrophosphate, together with earlier kinetic data, suggest a reaction mechanism where leucine, ATP and tRNA are bound to the enzyme in almost random order, and pyrophosphate is dissociated before the rate-limiting step. A kinetic analysis of this mechanism shows that the measured Kapp.i values do not give the real dissociation constant but it is about 0.4 mM. Other dissociation constants are 90 microM for leucine, 2.2 mM for ATP and 1 microM for tRNALeu. At the approximate conditions of the living cell (2 mM ATP, 100 microM leucine and 150 microM PPi) the leucyl-tRNA synthetase is about 85% inhibited by pyrophosphate.     

Enzymatic biosynthesis of ergotamine and investigation on some aminoacyl-tRNA synthetases in Claviceps

An enzyme system from Claviceps purpurea (Fr.) Tul. catalyzing the incorporation of l-phenylalanine into ergotamine - ergotamine synthetase - was purified 172-fold. This was done by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange chromatography on DEAE-Sepharose CL-6B, and hydroxyapatite chromatography. The activation of ergotamine specific amino acids as well as d-lysergic acid and dihydrolysergic acid via adenylates, as determined by the ATP-³²PP_i exchange, was investigated. Phenylalanyl-tRNA synthetase, catalyzing the same type of activation reaction, could not be separated from ergotamine synthetase by the purification procedure applied. Therefore, at the present stage of enzyme purification, phenylalanine-dependent ATP-³²PP_i exchange cannot be used to measure ergotamine synthetase activity specifically.Phenylalanyl-tRNA synthetase and leucyl-tRNA synthetase were separated into mitochondrial and cytoplasmic isoenzymes by hydroxyapatite chromatography. Their charging activities of procaryotic versus eucaryotic tRNA and their molecular masses were determined.