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.     

Mapping and cloning of Neurospora crassa mitochondrial transfer RNA genes.

We have obtained collections of recombinant Escherichia coli plasmids containing restriction fragments of Neurospora crassa mitochondrial DNA cloned into pBR322. By hybridization of 32P end-labeled total mitochondrial tRNAs and seven different purified tRNAs to restriction digests of mitochondrial DNA and of recombinant plasmids carrying specific restriction fragments, we have located the tRNA genes on the mitochondrial DNA. We have found that the mitochondrial tRNA genes are present in two major clusters, one between the two ribosomal RNA genes and the second closely following the large rRNA gene. Only one of the two DNA strands within these clusters codes for tRNAs. All of the genes for the seven specific purified tRNAs examined--those for alanine, formylmethionine, leucine 1, leucine 2, threonine, tyrosine, and valine--lie within these clusters. Interestingly, the formylmethionine tRNA hybridizes to two loci within one of these gene clusters. We have obtained a fairly detailed restriction map of part of this cluster and have shown that the two "putative" genes for formylmethionine tRNA are not arranged in tandem but are separated by more than 900 base pairs and by at least two other tRNA genes, those for alanine and for leucine 1 tRNAs.     

Transfer RNA genes in the cap-oxil region of yeast mitochondrial DNA.

A cytoplasmic "petite" (rho-) clone of Saccharomyces cerevisiae has been isolated and found through DNA sequencing to contain the genes for cysteine, histidine, leucine, glutamine, lysine, arginine, and glycine tRNAs. This clone, designated DS502, has a tandemly repeated 3.5 kb segment of the wild type genome from 0.7 to 5.6 units. All the tRNA genes are transcribed from the same strand of DNA in the direction cap to oxil. The mitochondrial DNA segment of DS502 fills a sequence gap that existed between the histidine and lysine tRNAs. The new sequence data has made it possible to assign accurate map positions to all the tRNA genes in the cap-oxil span of the yeast mitochondrial genome. A detailed restriction map of the region from 0 to 17 map units along with the locations of 16 tRNA genes have been determined. The secondary structures of the leucine and glutamine tRNAs have been deduced from their gene sequences. The leucine tRNA exhibits 64% sequence homology to an E. coli leucine tRNA.     

Transfer RNAs of Halobacterium volcanii: Sequences of five leucine and three serine tRNAs

The sequences of eight class II transfer RNAs (those having the large extra arm) of Halobacterium volcanii, five for leucine and three for serine are presented here. In principle, these tRNAs cover at least 11 out of the possible 12 codons for these two amino acids. Although these tRNAs follow general patterns for the class II tRNAs, in detail they are distinct from both eucaryotic and eubacterial tRNAs.     

Nematode-specific tRNAs that decode an alternative genetic code for leucine

Class II transfer RNAs (tRNAs), including tRNA(Leu) and tRNA(Ser), have an additional stem and loop structure, the long variable arm (V-arm). Here, we describe Class II tRNAs with a unique anticodon corresponding to neither leucine nor serine. Because these tRNAs are specifically conserved among the nematodes, we have called them 'nematode-specific V-arm-containing tRNAs' (nev-tRNAs). The expression of nev-tRNA genes in Caenorhabditis elegans was confirmed experimentally. A comparative sequence analysis suggested that the nev-tRNAs derived phylogenetically from tRNA(Leu). In vitro aminoacylation assays showed that nev-tRNA(Gly) and nev-tRNA(Ile) are only charged with leucine, which is inconsistent with their anticodons. Furthermore, the deletion and mutation of crucial determinants for leucylation in nev-tRNA led to a marked loss of activity. An in vitro translation analysis showed that nev-tRNA(Gly) decodes GGG as leucine instead of the universal glycine code, indicating that nev-tRNAs can be incorporated into ribosomes and participate in protein biosynthesis. Our findings provide the first example of unexpected tRNAs that do not consistently obey the general translation rules for higher eukaryotes.     

A reexamination of the leucine tRNAs and the leucyl-tRNA synthetase in developing Tenebrio molitor.

The translational control mechanism previously proposed for the synthesis of adult cuticular proteins in Tenebrio molitor is dependent upon the appearance of a major, novel leucine tRNA and a change in leucyl-tRNA synthetase activity just prior to adult emergence. The properties of the leucyl-tRNA synthetase extracted from pupae were reexamined. Under optimal aminoacylation conditions, no new leucine isoaccepting tRNAs were observed during development. However, under suboptimal conditions, a differential charging of the leucine tRNA species was noted. The chromatographic profiles of leucyl-tRNAs aminoacylated in vivo in both early and late pupae were found to be the same and were identical to the profiles obtained by charging tRNAs in vitro. Previous evidence for a translational control system operating in Tenebrio is discussed in relation to these results.     

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.     

Genetically determined differences in concentrations of isoaccepting tRNAs in Escherichia coli.

Two examples of genetically determined altered concentrations of isoaccepting tRNAs are presented. The concentrations of isoaccepting tRNAsThr are selectively changed by a mutation causing a fourfold overproduction of the cognate aminoacyl-tRNA-synthetase, the threonyl-tRNA synthetase, whereas the distribution of isoaccepting tRNAs of four control tRNA-species in these E. coli mutants was not affected by that mutation. Secondly evidence is presented for a correlation between mutations in structural genes of aminoacid biosynthetic enzymes and alterations in concentrations of cognate isoaccepting tRNAs in two different E. coli strains, auxotrophic for threonine, isoleucine/valine and leucine, and arginine respectively.     

Adaptation of the tRNA population of maize endosperm for zein synthesis.

Maize endosperm, 30 days after pollination is actively synthesizing zein, a storage protein containing high amounts of glutamine. leucine and alanine. Endosperm tRNAs have a higher accepting activity than embryo tRNAs for these three amino acids, but not for some other (control) amino acids. This increase in accepting activity is accompanied by a change in the distribution of the isoaccepting tRNA species corresponding to these three amino acids, but not of the isoacceptors corresponding to some other (control) amino acids. These results are in favor of the theory of functional adaptation of tRNA population.     

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.     

Modification defect at anticodon wobble nucleotide of mitochondrial tRNAs(Leu)(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes

The mitochondrial tRNA(Leu)(UUR) (R = A or G) gene possesses several hot spots for pathogenic mutations. A point mutation at nucleotide position 3243 or 3271 is associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes and maternally inherited diabetes with deafness. Detailed studies on two tRNAs(Leu)(UUR) with the 3243 or 3271 mutation revealed some common characteristics in cybrid cells: (i) a decreased life span, resulting in a 70% decrease in the amounts of the tRNAs in the steady state, (ii) a slight decrease in the ratios of aminoacyl-tRNAs(Leu)(UUR) versus uncharged tRNAs(Leu)(UUR), and (iii) accurate aminoacylation with leucine without any misacylation. As a marked result, both of the mutant tRNA molecules were deficient in a modification of uridine that occurs in the normal tRNA(Leu)(UUR) at the first position of the anticodon. The lack of this modification may lead to the mistranslation of leucine into non-cognate phenylalanine codons by mutant tRNAs(Leu)(UUR), according to the mitochondrial wobble rule, and/or a decrease in the rate of mitochondrial protein synthesis. This finding could explain why two different mutations (3243 and 3271) manifest indistinguishable clinical features.     

The ''polysemous'' codon--a codon with multiple amino acid assignment caused by dual specificity of tRNA identity.

In some Candida species, the universal CUG leucine codon is translated as serine. However, in most cases, the serine tRNAs responsible for this non-universal decoding (tRNA(Ser)CAG) accept in vitro not only serine, but also, to some extent, leucine. Nucleotide replacement experiments indicated that m1G37 is critical for leucylation activity. This finding was supported by the fact that the tRNA(Ser)CAGs possessing the leucylation activity always have m1G37, whereas that of Candida cylindracea, which possesses no leucylation activity, has A37. Quantification of defined aminoacetylated tRNAs in cells demonstrated that 3% of the tRNA(Ser)CAGs possessing m1G37 were, in fact, charged with leucine in vivo. A genetic approach using an auxotroph mutant of C.maltosa possessing this type of tRNA(Ser)CAG also suggested that the URA3 gene inactivated due to the translation of CUG as serine was rescued by a slight incorporation of leucine into the polypeptide, which demonstrated that the tRNA charged with multiple amino acids could participate in the translation. These findings provide the first evidence that two distinct amino acids are assigned by a single codon, which occurs naturally in the translation process of certain Candida species. We term this novel type of codon a 'polysemous codon'.     

Leucine transfer RNA from barley. Characterization and purification of isoaccepting species

Barley embryo leucine tRNA separated on reversed-phase chromatography-5 (RPC-5) into 5 fractions, whereas tRNA isolated from barley seedlings grown both in the light and in the dark, contained 4 species of tRNALeu. Species 2 and 3 were predominant; their relative ratio changed depending upon the growth conditions of the seedlings. Fractionation of crude barley tRNA successively on BD-cellulose, RPC-5 and Sepharose 4B enabled preparative isolation and purification of four leucine isoaccepting tRNAs. The species isolated differed in their main nucleotide composition, melting profiles and MgCl2 titration curves.     

Selective charging of tRNA isoacceptors induced by amino-acid starvation

Aminoacylated (charged) transfer RNA isoacceptors read different messenger RNA codons for the same amino acid. The concentration of an isoacceptor and its charged fraction are principal determinants of the translation rate of its codons. A recent theoretical model predicts that amino-acid starvation results in 'selective charging' where the charging levels of some tRNA isoacceptors will be low and those of others will remain high. Here, we developed a microarray for the analysis of charged fractions of tRNAs and measured charging for all Escherichia coli tRNAs before and during leucine, threonine or arginine starvation. Before starvation, most tRNAs were fully charged. During starvation, the isoacceptors in the leucine, threonine or arginine families showed selective charging when cells were starved for their cognate amino acid, directly confirming the theoretical prediction. Codons read by isoacceptors that retain high charging can be used for efficient translation of genes that are essential during amino-acid starvation. Selective charging can explain anomalous patterns of codon usage in the genes for different families of proteins.     

Mitochondrial tRNAs in the lower fungus Spizellomyces punctatus: tRNA editing and UAG 'stop' codons recognized as leucine.

The mitochondrial DNA of the chytridiomycete fungus Spizellomyces punctatusen codes only eight tRNAs, although a minimal set of 24-25 tRNAs is normally found in fungi. One of these tRNAs has a CAU anticodon and is structurally related to leucine tRNAs, which would permit the translation of the UAG 'stop' codons that occur in most of its protein genes. The predicted structures of all S. punctatus tRNAs have the common feature of containing one to three mis-pairings in the first three positions of their acceptor stems. Such mis-pairing is expected to impair proper folding and processing of tRNAs from their precursors. Five of these eight RNAs were shown to be edited at the RNA level, in the 5'portion of the molecules. These changes include both pyrimidine to purine and A to G substitutions that restore normal pairing in the acceptor stem. Editing was not found at other positions of the tRNAs, or in the mitochondrial mRNAs of S. punctatus. While tRNA editing has not been observed in other fungi, the editing pattern inS.punctatus is virtually identical to that described in the amoeboid protozoan Acanthamoeba castellanii. If this type of mitochondrial tRNA editing has originated from their common ancestor, one has to assume that it was independently lost in plants, animals and in most fungi. Alternatively, editing might have evolved independently, or the genes coding for the components of the editing machinery were laterally transferred.     

Role of taurine in the pathologies of MELAS and MERRF

Taurine is an abundant β-amino acid that concentrates in the mitochondria, where it participates in the conjugation of tRNAs for leucine, lysine, glutamate and glutamine. The formation of 5-taurinomethyluridine-tRNA strengthens the interaction of the anticodon with the codon, thereby promoting the decoding of several codons, including those for AAG, UUG, CAG and GAG. By preventing these series of events, taurine deficiency appears to diminish the formation of 5-taurinomethyluridine and causes inefficient decoding for the mitochondrial codons of leucine, lysine, glutamate and glutamine. The resulting reduction in the biosynthesis of mitochondria-encoded proteins deprives the respiratory chain of subunits required for the assembly of respiratory chain complexes. Hence, taurine deficiency is associated with a reduction in oxygen consumption, an elevation in glycolysis and lactate production and a decline in ATP production. A similar sequence of events takes place in mitochondrial diseases MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fiber syndrome). In both diseases, mutations in their respective tRNAs interfere with the formation of 5-taurinomethyluridine in the wobble position. Hence, the taurine-deficient phenotype resembles the phenotypes of MELAS and MERRF.     

The accuracy of poly(U) translation by different eukaryotic tRNAs

In this work we present comparative data on rates of phenylalanine and leucine incorporation into the poly(U) dependent product of cell-free translation by different eukaryotic tRNAs at high Mg2+ concentration. The frequency of translation errors has been found to depend upon the value of the tRNAPhe:tRNALeu ratio and the peculiarities of isoacceptor tRNAsLeu of different origin.     

An extra species of lysine transfer ribonucleic acid in polyoma virus-transformed cells in tissue culture

Isoaccepting tRNAs from various mouse cells were fractionated on columns of benzoylated DEAE cellulose. Lysine tRNA from mouse embryo, adult mouse liver and kidney, primary mouse embryo cells in tissue culture, and an established tissue culture line of mouse fibroblasts (3T3) has two peaks of isoaccepting tRNA; lysine tRNA from two established lines of polyoma virus-transformed cells contains an additional peak of lysine tRNA. The extra peak in transformed cells comprises about 25% of the acceptor capacity for lysine. It is stable to denaturation and renaturation and can be chromatographed, stripped of lysine, recharged, and rechromatographed. The extra peak is present in tRNA from transformed cells and absent in tRNA from normal cells regardless of whether the lysyl-tRNA ligase used for aminoacylation is from normal or transformed cells. Isoaccepting tRNAs for arginine, leucine, serine, valine, histidine, and tyrosine reveal similar profiles for the various tRNAs from normal and transformed cells.