Increased tRNA level in yeast cells with mutant translation termination factors eRF1 and eRF3

We have earlier characterized Saccharomyces cerevisiae strains with mutations of essential SUP45 and SUP35, which code for translation termination factors eRF1 and eRF3, respectively. In this work, the sup45 and sup35 nonsense mutants were compared with respect to the levels of eight tRNAs: tRNA^Tyr, tRNA^Gln, tRNA^Trp, tRNA^Leu, tRNA^Arg (described as potential suppressor tRNAs), tRNA^Pro, tRNA^His, and tRNA^Gly. The mutants did not display a selective increase in tRNAs, capable of a noncanonical read-through at stop codons. Most of the mutations increased the level of all tRNAs under study. The mechanisms providing for the viability of the sup45 and sup35 nonsense mutants are discussed.     

Glycine and asparagme tRNA sequences from the archaebacteriuin,Methanobacterium thermoautotrophicum

Two tRNA sequences from Methanobacterium thermoautotrophium are reported. Both tRNA^Gly_GCC and tRNA_NUU^Asn, the first tRNA sequences from methanogens, were determined by partial hydrolyses (both chemical and enzymatic) and analyzed by gel electrophoresis. The two tRNAs contain the unusual T-loop modifications, Cm and m¹I, which are present in other archaebacterial tRNAs. Finally the presence of an unknown modification in the D-loop has been inferred by a large jump in the sequence ladder. These tRNAs are approximately equidistant from eubacterial or eukaryotic tRNAs.     

Transfer RNA gene mapping studies on chloroplast DNA from Chlamydomonas reinhardii

Total tRNA of Chlamydomonas reinhardii was fractionated by 2-dimensional gel electrophoresis. Sixteen tRNAs specific for eleven amino acids could be identified by aminoacylation with Escherichia coli tRNA synthetases. Hybridization of these tRNAs with chloroplast restriction fragments allowed for the localization of the genes of tRNA^Tyr, tRNA^Pro, tRNA^Phe (2 genes), tRNA^Ile (2 genes) and tRNA^His (2 genes) on the chloroplast genome of C. reinhardii. The genes for tRNA^Ala (2 genes), tRNA^Asn and tRNA^Leu were mapped by using individual chloroplast tRNAs from higher plants as probes.     

Striking differences in mitochondrial tRNA import between different plant species

A systematic comparison of the tRNAs imported into the mitochondria of larch, maize and potato reveals considerable differences among the three species. Larch mitochondria import at least eleven different tRNAs (more than half of those tested) corresponding to ten different amino acids. For five of these tRNAs [tRNA^Phe(GAA), tRNA^Lys(CUU), tRNA^Pro(UGG), tRNA^Ser(GCU) and tRNA^Ser(UGA)] this is the first report of import into mitochondria in any plant species. There are also differences in import between relatively closely related plants; wheat mitochondria, unlike maize mitochondria import tRNA^His, and sunflower mitochondria, unlike mitochondria from other angiosperms tested, import tRNA^Ser(GCU) and tRNA^Ser(UGA). These results suggest that the ability to import each tRNA has been acquired independently at different times during the evolution of higher plants, and that there are few apparent restrictions on which tRNAs can or cannot be imported. The implications for the mechanisms of mitochondrial tRNA Import in plants are discussed.     

Studies on the ribothymidine content of specific rat and human tRNAs: a postulated role for 5-methyl cytosine in the regulation of ribothymidine biosynthesis.

Total mammalian tRNAs contain on the average less than one mole of ribothymidine per mole of tRNA. Mammalian tRNAs can be grouped into at least four classes, depending upon their ribothymidine content at position 23 from the 3′ terminus. Class A contains tRNA in which a nucleoside other than uridine replaces ribothymidine (tRNA_i^Met); Class B contains tRNA in which one mole of a modified uridine (rT, ψ, or 2′-O-methylribothymidine) is found per mole of tRNA (tRNA^Ser, tRNA^Trp, and tRNA^Lys, respectively). Class C contains tRNA in which there is a partial conversion of uridine to ribothymidine (tRNA^Phe, tRNA₁^Gly, tRNA₂^Gly); Class D contains tRNA which totally lacks ribothymidine (tRNA^Val). Only those tRNAs in Class C are acceptable substrates for $\text{E.}\text{coli}$ uridine methylase, under the conditions used in these studies. These observations cannot be adequately explained solely on the basis of the presence or absence of a specific “universal” nucleoside other than U or rT at position 23 from the 3′ terminus. However, correlations can be made between the ribothymidine and 5-methylcytosine content of eucaryotic tRNA. We postulate that the presence of one or more 5-methylcytosines in and adjacent to loop III (minor loop) in individual tRNAs act to regulate the amount of ribothymidine formed by uridine methylase. Several experiments are proposed as tests for this hypothesis.     

On the Possible Role of tRNA Base Modifications in the Evolution of Codon Usage: Queuosine and Drosophila

The best documented selection-based hypothesis to explain unequal usage of codons is based on the relative abundance of isoaccepting tRNAs. In unicellular organisms the most used codons are optimally translated by the most abundant tRNAs. The chemical bonding energies are affected by modification of the four traditional bases, in particular in the first anti-codon corresponding to the third codon position. One nearly universal modification is queuosine (Q) for guanine (G) in tRNA^His, tRNA^Asp, tRNA^Asn, and tRNA^Tyr; this changes the optimal binding from codons ending in C to no preference or a slight preference for U-ending codons. Among species of Drosophila, codon usage is constant with the exception of the Drosophila willistoni lineage which has shifted primary usage from C-ending codons to U/T ending codons only for these four amino acids. In Drosophila melanogaster Q containing tRNAs only predominate in old adults. We asked the question whether in D. willistoni these Q containing tRNAs might predominate earlier in development. As a surrogate for levels of modification we studied the expression of the gene (tgt) coding for the enzyme that catalyzes the substitution of Q for G in different life stages of D. melanogaster, D. pseudoobscura, and D. willistoni. Unlike the other two species, the highest tgt expression in D. willistoni is in young females producing eggs. Because tRNAs laid down in eggs persist through the early stages of development, this implies that Q modification occurs earlier in development in D. willistoni than in other Drosophila.     

The output of the tRNA modification pathways controlled by the Escherichia coli MnmEG and MnmC enzymes depends on the growth conditions and the tRNA species

In Escherichia coli, the MnmEG complex modifies transfer RNAs (tRNAs) decoding NNA/NNG codons. MnmEG catalyzes two different modification reactions, which add an aminomethyl (nm) or carboxymethylaminomethyl (cmnm) group to position 5 of the anticodon wobble uridine using ammonium or glycine, respectively. In and , however, cmnm⁵ appears as the final modification, whereas in the remaining tRNAs, the MnmEG products are converted into 5-methylaminomethyl (mnm⁵) through the two-domain, bi-functional enzyme MnmC. MnmC(o) transforms cmnm⁵ into nm⁵, whereas MnmC(m) converts nm⁵ into mnm⁵, thus producing an atypical network of modification pathways. We investigate the activities and tRNA specificity of MnmEG and the MnmC domains, the ability of tRNAs to follow the ammonium or glycine pathway and the effect of mnmC mutations on growth. We demonstrate that the two MnmC domains function independently of each other and that and are substrates for MnmC(m), but not MnmC(o). Synthesis of mnm⁵s²U by MnmEG-MnmC in vivo avoids build-up of intermediates in . We also show that MnmEG can modify all the tRNAs via the ammonium pathway. Strikingly, the net output of the MnmEG pathways in vivo depends on growth conditions and tRNA species. Loss of any MnmC activity has a biological cost under specific conditions.     

Ubiquity of selenium-containing tRNA in plants

Sodium[⁷⁵Se]selenite supplemented culture of Chlamydomonas, wild carrot, tobacco, bamboo, and rice cells as well as mung bean and soybean seedlings incorporated, without exception, ⁷⁵Se into tRNAs. The content of ⁷⁵Se-labeled tRNAs ranged from 0.04 to 1.89% of the total tRNAs in these seven plant species. [⁷⁵Se]tRNA samples of wild carrot and mung bean were fractionated into six or seven seleno-tRNA species by chromatography on RPC-5 column. Samples of tobacco, bamboo and Chlamydomonas each exhibited only a single seleno-tRNA species with a close interspecific resemblance in the elution position among the three samples. All these [⁷⁵Se]tRNAs contained a new, not yet identified ⁷⁵Se-labeled nucleoside, whose retention time on HPLC was distinctly different from that of the previously reported bacterial selenonucleosides. [⁷⁵Se]tRNA samples of rice, tobacco, bamboo, mung bean and Chlamydomonas also contained one or two minor ⁷⁵Se-labeled nucleosides. These results suggest that (1) selenium-containing tRNAs appear to be widespread in the plant kingdom and (2) a new, not yet characterized selenonucleoside might be universal in plants.     

The tRNA methyltransferase Dnmt2 is required for accurate polypeptide synthesis during haematopoiesis

The Dnmt2 enzyme utilizes the catalytic mechanism of eukaryotic DNA methyltransferases to methylate several tRNAs at cytosine 38. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile, and the biological function of Dnmt2 has remained elusive. Here, we show that endochondral ossification is delayed in newborn Dnmt2‐deficient mice, which is accompanied by a reduction of the haematopoietic stem and progenitor cell population and a cell‐autonomous defect in their differentiation. RNA bisulfite sequencing revealed that Dnmt2 methylates C38 of tRNA Asp^GTC, Gly^GCC, and Val^AAC, thus preventing tRNA fragmentation. Proteomic analyses from primary bone marrow cells uncovered systematic differences in protein expression that are due to specific codon mistranslation by tRNAs lacking Dnmt2‐dependent methylation. Our observations demonstrate that Dnmt2 plays an important role in haematopoiesis and define a novel function of C38 tRNA methylation in the discrimination of near‐cognate codons, thereby ensuring accurate polypeptide synthesis.     

Conformational peculiarities of rRNA inff supMet from E. coli as revealed by fluorescent methods

Conformational transitions in several individual tRNAs (tRNA _inff ^supMet , tRNA^Phe from E. coli, tRNA _inf1 ^supVal , tRNA^Ser, tRNA^Phe from yeast) have been studied under various environmental conditions. The binding isotherms studies for dyes-tRNA complexes exhibited similarities in conformational states of all tRNAs investigated at low ionic strength (0.01 M NaCl). By contrast, at high ionic strength (0.4 M NaCl or 2×10^-4 M Mg²⁺) a marked difference is found in structural features of tRNA _inff ^supMet as compared with other tRNAs used. The tRNA _inff ^supMet is the only tRNA species that does not reveal the strong type of complexes with ethidium bromide, acriflavine and acridine orange.     

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.     

Thiolated tRNAs of Trypanosoma brucei Are Imported into Mitochondria and Dethiolated after Import

All mitochondrial tRNAs in Trypanosoma brucei derive from cytosolic tRNAs that are in part imported into mitochondria. Some trypanosomal tRNAs are thiolated in a compartment-specific manner. We have identified three proteins required for the thio modification of cytosolic tRNA^Gln, tRNA^Glu, and tRNA^Lys. RNA interference-mediated ablation of these proteins results in the cytosolic accumulation non-thio-modified tRNAs but does not increase their import. Moreover, in vitro import experiments showed that both thio-modified and non-thio-modified tRNA^Glu can efficiently be imported into mitochondria. These results indicate that unlike previously suggested the cytosol-specific thio modifications do not function as antideterminants for mitochondrial tRNA import. Consistent with these results we showed by using inducible expression of a tagged tRNA^Glu that it is mainly the thiolated form that is imported in vivo. Unexpectedly, the imported tRNA becomes dethiolated after import, which explains why the non-thiolated form is enriched in mitochondria. Finally, we have identified two genes required for thiolation of imported tRNA^Trp whose wobble nucleotide is subject to mitochondrial C to U editing. Interestingly, down-regulation of thiolation resulted in an increase of edited tRNA^Trp but did not affect growth.     

Increased activity of rat liver N2-guanine tRNA methyltransferase II in response to liver damage

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5′ end of the molecule. This group of tRNAs includes E. coli tRNA^Nfmet, tRNA^Ala₁, tRNA^Leu₁, or ^Leu₂, and tRNA^Ser₃ (Group 1). In each case N²-methylguanine and N²,N²-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N²-guanine methyltransferase II ($\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine:tRNA}$ (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50°C for 10 min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preperations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N²-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.     

Mature methyl-deficient tRNA isolated from a mammary adenocarcinoma

A transplantable rat tumor, mammary adenocarcinoma 13762, accumulates tRNA which can be methylated in vitro by mammalian tRNA (adenine-1) methyltransferase. This unusual ability of the tumor RNA to serve as substrate for a homologous tRNA methylating enzyme is correlated with unusually low levels of the A₅₈-specific adenine-1 methyltransferase. The nature of the methyl-accepting RNA has been examined by separating tumor tRNA on two-dimensional polyacrylamide gels. Comparisons of ethidium bromide-stained gels of tumor vs. liver tRNA show no significant quantitative differences and no accumulation of novel tRNAs or precursor tRNAs in adenocarcinoma RNA. Two-dimensional separations of tumor RNA after in vitro [¹⁴C]methylation using purified adenine-1 methyltransferase indicate that about 25% of the tRNA species are strongly methyl-accepting RNAs. Identification of six of the tRNAs separated on two-dimensional gels has been carried out by hybridization of cloned tRNA genes to Northern blots. Three of these, tRNA^Lys3, tRNA^Gln and tRNA^Met_i, are among the adenocarcinoma methyl-accepting RNAs. The other three RNAs, all of which are leucine-specific tRNAs, show no methyl-accepting properties. Our results suggest that low levels of a tRNA methyltransferase in the adenocarcinoma cause selected species of tRNA to escape the normal A₅₈ methylation, resulting in the appearance of several mature tRNAs which are deficient in 1-methyladenine. The methyl-accepting tRNAs from the tumor appear as ethidium bromide-stained spots of similar intensity to those seen for RNA from rat liver; therefore, methyladenine deficiency does not seem to impair processing of these tRNAs.     

Sequence Relationship of Three Valine Acceptor tRNAs from <Emphasis Type="Italic">Escherichia coli</Emphasis>

THE degree of degeneracy of the genetic code varies for the twenty amino-acids: between one and six different triplets are assigned to a single amino-acid. Four triplets GUU, GUC, GUA, GUG code for the amino-acid valine^1,2. Two valine specific tRNAs have been separated by fractionation of mixed E. coli tRNA³; tRNA^val₁ is specific for GU^A_G and tRNA^val₂ corresponds to GU^U_C (see also ref. 1 for binding properties). Recent studies showed that although both species are recognized by the single activating enzyme present in E. coli, the association constant (Ka) for the minor species, tRNA^val₂ (?20% of total acceptor), is an order of magnitude higher than the association constant of the major species, tRNA^{val 4}₁. As a first step to comparing the structures of these two tRNAs, we analysed the base sequences of the major and minor species. We recently published the nucleotide sequence of tRNA^{val 5}₁; we report here the sequence of two minor subspecies (quite similar to each other) that comprise the tRNA^val₂ acceptor and we comment on the significance of the sequence homologies in relation to the problems of enzyme recognition and tRNA evolution.     

Queuosine deficient tRNAHis and tRNAAsp from the spleens of young mice,erythroleukemic tumoral spleens and cultured Friend cells

tRNAs were isolated from the spleens of young mice, erythroleukemic spleens and cultured Friend cells. Queuosine (Q) deficient tRNAs were labelled in their anticodon with radioactive guanine using the exchange reaction catalyzed by E. coli tRNA-guanine transglycosylase. tRNAs were then specifically aminoacylated. In both normal and tumoral spleen (TF-P10), tRNA^His was the main guanine containing (G) isoacceptor as shown by RPC-5 chromatography. In vitro, the tumor derived cell line (TF-P10c) retained the G-tRNA^His species while Friend cell line, clone 707 (FLC), did not. A common feature found in both cultured cell lines was the high level of G-tRNA^Asp. The meaning of the relative abundance of Q-deficient tRNA^His and of tRNA^Asp observed in transformed cells of erythroid origin is discussed.     

Conformational changes in the thiouridine region of Escherichia coli transfer RNA as assessed by photochemically induced crosslinking: Effect of a single base change in the “extra” loop of formylmethionine tRNA and of denaturation of tryptophan tRNA

Photochemical crosslinking studies on two formylmethionine tRNAs of Escherichia coli are consistent with the hypothesis that the role of 7-methylguanosine is to stabilize a tertiary structure of tRNA in which the “extra” loop is folded over so as to be close to the 4-thiouridine region of the molecule. In tRNA^{fmet 3}, which differs from tRNA^{fmet 1} only by substitution of an adenosine for the 7-methylguanosine in the “extra” loop, crosslinking was virtually abolished when the tRNA was placed in 40 mm Na⁺, whereas tRNA^{fmet 1} in 40 mm Na⁺ was crosslinked to 95% of the maximum extent observed for both tRNAs in Mg²⁺. Even in Mg²⁺, a difference in structure between the two tRNAs could be detected by means of a two-fold decrease in the rate of crosslinking in tRNA^{fmet 3} as compared to tRNA^{fmet 1}. Comparison of crosslinking in the native and metastable denatured forms of tRNA^Trp of E. coli revealed that these structures also differ with respect to the orientation and/or distance between 4-thiouridine (8) and cytidine (13), since denaturation abolished crosslinking. However, separation of these two residues is not obligatory for denaturation, since crosslinked tRNA^Trp could still be denatured. A 30% difference in fluorescence between the native and denatured forms of crosslinked-reduced tRNA^Trp infers an increase in hydrophilicity in the 4-thiouridine region upon denaturation.     

Partition of tRNAGly isoacceptors between protein and cell-wall peptidoglycan synthesis in Staphylococcus aureus

The sequence of tRNAs is submitted to evolutionary constraints imposed by their multiple interactions with aminoacyl-tRNA synthetases, translation elongation factor Tu in complex with GTP (EF-Tu•GTP), and the ribosome, each being essential for accurate and effective decoding of messenger RNAs. In Staphylococcus aureus, an additional constraint is imposed by the participation of tRNA^Gly isoacceptors in the addition of a pentaglycine side chain to cell-wall peptidoglycan precursors by transferases FmhB, FemA and FemB. Three tRNA^Gly isoacceptors poorly interacting with EF-Tu•GTP and the ribosome were previously identified. Here, we show that these ‘non-proteogenic’ tRNAs are preferentially recognized by FmhB based on kinetic analyses and on synthesis of stable aminoacyl-tRNA analogues acting as inhibitors. Synthesis of chimeric tRNAs and of helices mimicking the tRNA acceptor arms revealed that this discrimination involves identity determinants exclusively present in the D and T stems and loops of non-proteogenic tRNAs, which belong to an evolutionary lineage only present in the staphylococci. EF-Tu•GTP competitively inhibited FmhB by sequestration of ‘proteogenic’ aminoacyl-tRNAs in vitro. Together, these results indicate that competition for the Gly-tRNA^Gly pool is restricted by both limited recognition of non-proteogenic tRNAs by EF-Tu•GTP and limited recognition of proteogenic tRNAs by FmhB.