Extensive tRNA Gene Changes in Synthetic Brassica napus

Allopolyploidization, where two species come together to form a new species, plays a major role in speciation and genome evolution. Transfer RNAs (abbreviated tRNA) are typically 73–94 nucleotides in length, and are indispensable in protein synthesis, transferring amino acids to the cell protein synthesis machinery (ribosome). To date, the regularity and function of tRNA gene sequence variation during the process of allopolyploidization have not been well understood. In this study, the inter-tRNA gene corresponding to tRNA amplification polymorphism method was used to detect changes in tRNA gene sequences in the progeny of interspecific hybrids between Brassica rapa and B. oleracea, mimicking the original B. napus (canola) species formation event. Cluster analysis showed that tRNA gene variation during allopolyploidization did not appear to have a genotypic basis. Significant variation occurred in the early generations of synthetic B. napus (F₁ and F₂ generations), but fewer alterations were observed in the later generation (F₃). The variation-prone tRNA genes tended to be located in AT-rich regions. BlastN analysis of novel tRNA gene variants against a Brassica genome sequence database showed that the variation of these tRNA-gene-associated sequences in allopolyploidization might result in variation of gene structure and function, e.g., metabolic process and transport.     

Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10

N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m¹G₉ modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m¹G₉ in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m¹G₉ methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m¹G₉ containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.     

Isolation of transfer RNA isoacceptors by chromatography on dihydroxyboryl-substituted cellulose,polyacrylamide, and glass

Polyacrylamide and porous-glass supports containing the dihydroxyborylphenyl group can be prepared by a method similar to that used in the synthesis of N-[N′-(m-dihydroxyborylphenyl)succinamyl]aminoethylcellulose. The reaction of aminoethylpolyacrylamide or amino-substituted glass with N-(m-dihydroxyborylphenyl)succinamic acid in the presence of N-cyclohexyl-N′-β-(4-methyl-morpholinium) ethylcarbodiimide yields products which, together with the cellulose derivative, are all capable of binding tRNA dissolved in buffers at pH 8.7. The demonstration that bound tRNA can be released with sorbitol solutions or with low pH buffers, together with studies on the binding of tRNA species that contain chemically modified 3′-terminals, indicate that the predominant binding mechanism consists of cyclic complex formation between the immobilized dihydroxyboryl groups and the 3′-terminal cis-diol groups of the tRNA molecules. Aminoacylated tRNA does not bind under the conditions necessary to bind tRNA and this permits the isolation of specific tRNA isoacceptors. The purification of tRNA^Phe and the partial purification of tRNA^Glu and tRNA^Trp are described.     

Identification of Japanese Lymantria species (Lepidoptera: Lymantriidae) based on PCR–RFLP analysis of mitochondrial DNA

A polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP)-based method for species identification was applied to seven Japanese Lymantria species, including four Asian gypsy moth (AGM) species. We sequenced the partial end of the cytochrome c oxidase I (COI) gene, tRNA leucine, COII gene, and partial end of the tRNA lysine in mitochondrial DNA (mtDNA) for one individual of each of the seven species. We analyzed the recognition sites of three restriction endonucleases and constructed a scheme for Lymantria species identification using PCR–RFLP. We then applied the scheme to 291 individuals from 45 populations of seven species. We found that all seven species were correctly identified using PCR–RFLP. These results suggest that PCR–RFLP is useful for identifying Japanese Lymantria species, which may be detected at Japanese ports.     

Diverse cell stresses induce unique patterns of tRNA up- and down-regulation: tRNA-seq for quantifying changes in tRNA copy number

Emerging evidence points to roles for tRNA modifications and tRNA abundance in cellular stress responses. While isolated instances of stress-induced tRNA degradation have been reported, we sought to assess the effects of stress on tRNA levels at a systems level. To this end, we developed a next-generation sequencing method that exploits the paucity of ribonucleoside modifications at the 3′-end of tRNAs to quantify changes in all cellular tRNA molecules. Application of this tRNA-seq method to Saccharomyces cerevisiae identified all 76 expressed unique tRNA species out of 295 coded in the yeast genome, including all isoacceptor variants, with highly precise relative (fold-change) quantification of tRNAs. In studies of stress-induced changes in tRNA levels, we found that oxidation (H₂O₂) and alkylation (methylmethane sulfonate, MMS) stresses induced nearly identical patterns of up- and down-regulation for 58 tRNAs. However, 18 tRNAs showed opposing changes for the stresses, which parallels our observation of signature reprogramming of tRNA modifications caused by H₂O₂ and MMS. Further, stress-induced degradation was limited to only a small proportion of a few tRNA species. With tRNA-seq applicable to any organism, these results suggest that translational control of stress response involves a contribution from tRNA abundance.     

Protein Synthesis in E. coli: Dependence of Codon-Specific Elongation on tRNA Concentration and Codon Usage

To synthesize a protein, a ribosome moves along a messenger RNA (mRNA), reads it codon by codon, and takes up the corresponding ternary complexes which consist of aminoacylated transfer RNAs (aa-tRNAs), elongation factor Tu (EF-Tu), and GTP. During this process of translation elongation, the ribosome proceeds with a codon-specific rate. Here, we present a general theoretical framework to calculate codon-specific elongation rates and error frequencies based on tRNA concentrations and codon usages. Our theory takes three important aspects of in-vivo translation elongation into account. First, non-cognate, near-cognate and cognate ternary complexes compete for the binding sites on the ribosomes. Second, the corresponding binding rates are determined by the concentrations of free ternary complexes, which must be distinguished from the total tRNA concentrations as measured in vivo. Third, for each tRNA species, the difference between total tRNA and ternary complex concentration depends on the codon usages of the corresponding cognate and near-cognate codons. Furthermore, we apply our theory to two alternative pathways for tRNA release from the ribosomal E site and show how the mechanism of tRNA release influences the concentrations of free ternary complexes and thus the codon-specific elongation rates. Using a recently introduced method to determine kinetic rates of in-vivo translation from in-vitro data, we compute elongation rates for all codons in Escherichia coli. We show that for some tRNA species only a few tRNA molecules are part of ternary complexes and, thus, available for the translating ribosomes. In addition, we find that codon-specific elongation rates strongly depend on the overall codon usage in the cell, which could be altered experimentally by overexpression of individual genes.     

Three Different Missense Suppressor Mutations Affecting the tRNAGGGGly Species of Escherichia coli

The Escherichia coli suppressor mutation, supT, has been shown to cause a C → U substitution in the middle position of the tRNA_GGG^Gly anticodon. This is the same tRNA species that is altered by the glyUsu_AGA mutation studied previously. This finding indicates that the supT mutant tRNA reads the glutamic acid codon, GAG. The supT suppressor has also been converted to a new suppressor, called glyUsu_GAA, which will suppress the GAA mutation, trpA46. The in vivo suppression efficiencies of each of these three missense suppressors has been measured and are as follows: glyUsu_AGA, 3.6%; supT, 1.6%; and glyUsu_GAA, 0.4%. Mistranslation by these mutant glycine tRNA species has no adverse affects on cell growth since cultures possessing the suppressors grow as fast as cells without. The supT tRNA species can be observed as a peak in the profile of glycyl-tRNA fractionated on a RPC-5 chromatographic column, indicating that the mutant tRNA can be aminoacylated with reasonable efficiency. This finding contrasts with previous findings concerning the glyUsu_AGA mutant tRNA which is not significantly aminoacylated under the same conditions.     

Cytokinin-Active Ribonucleosides in Phaseolus RNA: II. DISTRIBUTION IN tRNA SPECIES FROM ETIOLATED P. VULGARIS L. SEEDLINGS

The distribution of cytokinin-active ribonucleosides in tRNA species from etiolated Phaseolus vulgaris L. seedlings has been examined. Phaseolus tRNA was fractionated by benzoylated diethylaminoethyl-cellulose and RPC-5 chromatography, and the distribution of cytokinin activity was compared with the distribution of tRNA species expected to correspond to codons beginning with U. Phaseolus tRNA^Cys, tRNA^Trp, tRNA^Tyr, a major peak of tRNA^Phe, and a large fraction of tRNA^Leu were devoid of cytokinin activity in the tobacco bioassay. Cytokinin activity was associated with all fractions containing tRNA^Ser species and with minor tRNA^Leu species. In addition, several anomalous peaks of cytokinin activity that could not be directly attributed to U group tRNA species were detected.     

Purification of Leucine tRNA Isoaccepting Species from Soybean Cotyledons: II. RPC-2 Purification, Ribosome Binding, and Cytokinin Content

Two of the six leucine isoaccepting tRNA species from soybean (Glycine max) cotyledons recognize U-beginning codons, and contain cytokinin moieties in their structure. These same two isoaccepting species have been shown to undergo quantitative changes in their relative amounts upon treatment with N⁶-benzyladenine in vivo. In addition a procedure has been developed for purification of the isoaccepting species of leucine tRNA from soybean cotyledons resulting in isoacceptors of minimum purity, calculated by amino acid acceptance capacity, of from 46 to 78% leucine tRNA.     

A variable automatic recording device for measuring phytochrome with the Perkin-Elmer spectrophotometer model 356

A method is described for the quantitative analysis and preparative isolation of N-[N-methyl-N-(9-β-ribofuranosylpurin-6-yl)carbamoyl]threonine (mt⁶A), a rare modified nucleoside constituent of transfer RNA. This method is based on the selective retention of mt⁶A and its parent compound, t⁶A, on Dowex-1 at pH 7.8, allowing these two nucleosides to be readily concentrated from the mixture of nucleosides resulting when tRNA is hydrolyzed by a combination of snake venom enzymes and E. coli alkaline phosphatase. The content of mt⁶A in wheat embryo and E. coli tRNA was found to be about 0.025 mole %, which is roughly one-tenth the t⁶A content of the tRNA of these two organisms. Since this indicates that only about 1 in 50 chains can contain a residue of mt⁶A, this nucleoside may be confined to a single isoaccepting species of transfer RNA in both E. coli and wheat embryo. No mt⁶A could be detected in either baker's or brewer's yeast tRNA by the method described. Either mt⁶A is entirely absent from yeast tRNA or it occurs in a form which does not adsorb to Dowex-1 during fractionation of hydrolysates of yeast tRNA. No t⁶A or mt⁶A could be detected in the 18 S + 26 S ribosomal ribonucleates of wheat embryo.     

Loss of a Conserved tRNA Anticodon Modification Perturbs Plant Immunity

tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined. Despite their vastly different chemical structures and their presence in different tRNAs, occurring in different locations in tRNA, the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent discoveries have revealed unprecedented complexity in the modification patterns of tRNA, their regulation and function, suggesting that each modified nucleoside in tRNA may have its own specific function. However, in plants, our knowledge on the role of individual tRNA modifications and how they are regulated is very limited. In a genetic screen designed to identify factors regulating disease resistance and activation of defenses in Arabidopsis, we identified SUPPRESSOR OF CSB3 9 (SCS9). Our results reveal SCS9 encodes a tRNA methyltransferase that mediates the 2´-O-ribose methylation of selected tRNA species in the anticodon loop. These SCS9-mediated tRNA modifications enhance during the course of infection with the bacterial pathogen Pseudomonas syringae DC3000, and lack of such tRNA modification, as observed in scs9 mutants, severely compromise plant immunity against the same pathogen without affecting the salicylic acid (SA) signaling pathway which regulates plant immune responses. Our results support a model that gives importance to the control of certain tRNA modifications for mounting an effective immune response in Arabidopsis, and therefore expands the repertoire of molecular components essential for an efficient disease resistance response.     

Distribution of Cytokinin-active Ribonucleosides in Wheat Germ tRNA Species

The distribution of cytokinin activity in wheat (Triticum aestivum) germ tRNA fractionated by BD-cellulose and RPC-5 chromatography has been examined. As in other organisms, the cytokinin moieties in wheat germ tRNA appear to be restricted to tRNA species that would be expected to respond to codons beginning with U. Only a few of the wheat germ tRNA species in this coding group actually contain cytokinin modifications. Cytokinin activity was associated with isoaccepting tRNA^Ser species and with a minor tRNA^Leu species from wheat germ. All other wheat germ tRNA species corresponding to codons beginning with U were devoid of cytokinin activity in the tobacco callus bioassay.     

An evolutionary approach uncovers a diverse response of tRNA 2-thiolation to elevated temperatures in yeast

Chemical modifications of transfer RNA (tRNA) molecules are evolutionarily well conserved and critical for translation and tRNA structure. Little is known how these nucleoside modifications respond to physiological stress. Using mass spectrometry and complementary methods, we defined tRNA modification levels in six yeast species in response to elevated temperatures. We show that 2-thiolation of uridine at position 34 (s²U₃₄) is impaired at temperatures exceeding 30°C in the commonly used Saccharomyces cerevisiae laboratory strains S288C and W303, and in Saccharomyces bayanus. Upon stress relief, thiolation levels recover and we find no evidence that modified tRNA or s²U₃₄ nucleosides are actively removed. Our results suggest that loss of 2-thiolation follows accumulation of newly synthesized tRNA that lack s²U₃₄ modification due to temperature sensitivity of the URM1 pathway in S. cerevisiae and S. bayanus. Furthermore, our analysis of the tRNA modification pattern in selected yeast species revealed two alternative phenotypes. Most strains moderately increase their tRNA modification levels in response to heat, possibly constituting a common adaptation to high temperatures. However, an overall reduction of nucleoside modifications was observed exclusively in S288C. This surprising finding emphasizes the importance of studies that utilize the power of evolutionary biology, and highlights the need for future systematic studies on tRNA modifications in additional model organisms.     

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.     

The Catalytic Domain of Topological Knot tRNA Methyltransferase (TrmH) Discriminates between Substrate tRNA and Nonsubstrate tRNA via an Induced-fit Process

A conserved guanosine at position 18 (G18) in the D-loop of tRNAs is often modified to 2′-O-methylguanosine (Gm). Formation of Gm18 in eubacterial tRNA is catalyzed by tRNA (Gm18) methyltransferase (TrmH). TrmH enzymes can be divided into two types based on their substrate tRNA specificity. Type I TrmH, including Thermus thermophilus TrmH, can modify all tRNA species, whereas type II TrmH, for example Escherichia coli TrmH, modifies only a subset of tRNA species. Our previous crystal study showed that T. thermophilus TrmH is a class IV S-adenosyl-l-methionine-dependent methyltransferase, which maintains a topological knot structure in the catalytic domain. Because TrmH enzymes have short stretches at the N and C termini instead of a clear RNA binding domain, these stretches are believed to be involved in tRNA recognition. In this study, we demonstrate by site-directed mutagenesis that both N- and C-terminal regions function in tRNA binding. However, in vitro and in vivo chimera protein studies, in which four chimeric proteins of type I and II TrmHs were used, demonstrated that the catalytic domain discriminates substrate tRNAs from nonsubstrate tRNAs. Thus, the N- and C-terminal regions do not function in the substrate tRNA discrimination process. Pre-steady state analysis of complex formation between mutant TrmH proteins and tRNA by stopped-flow fluorescence measurement revealed that the C-terminal region works in the initial binding process, in which nonsubstrate tRNA is not excluded, and that structural movement of the motif 2 region of the catalytic domain in an induced-fit process is involved in substrate tRNA discrimination.     

An RNA species involved in Escherichia coli ribonuclease P activity. Gene cloning and effect on transfer RnA synthesis in vivo

The isolation and characterization of a plasmid capable of complementing the temperature-sensitive transfer RNA biosynthetic mutation rnpA49 (ribonuclease P) is described. The DNA segment responsible for complementation codes for an RNA species, approximately 340 bases long. Hybridization-selection experiments indicated that all rnp mutants were deficient in the production of the complementing RNA at high temperature; these defects were not due to the accumulation of a precursor form of this RNA. Examination of tRNA species synthesized in vivo indicated that the plasmid clone did not completely relieve the deficiency in RNase P activity of rnpA49 since some tRNA precursors still accumulated in strain A49 containing the plasmid. At least one other tRNA precursor was no longer detectable in plasmid-containing rnpA49 cells.     

Discrimination between aminoacyl groups on su+ 7 tRNA by elongation factor Tu

The su⁺7 nonsense suppressor of Escherichia coli is a mutant tRNA^Trp that can be aminoacylated with either tryptophan or glutamine. We have compared the ternary complexes of glutaminyl and tryptophanyl-su⁺7 tRNA with elongation factor Tu and GTP. Glutaminyl-su⁺7 tRNA binds more strongly than tryptophanyl-su⁺7 tRNA to EF Tu · GTP. The greatest distinction between the two species of the tRNA is seen in their dissociation rates from the complex, which differ by as much as fivefold. The distinction is affected by pH values around neutrality. These results show that EF Tu can distinguish between two aminoacyl-tRNAs which differ only in the aminoacyl group. The implications for the unusual amino acid specificity of su⁺7 tRNA are discussed.     

A novel suppressor tRNA from the dimorphic fungus Candida albicans

Unfractionated tRNAs from a number of prokaryotes and eukaryotes were examined for their ability to promote termination codon readthrough in a cell-free system isolated from Saccharomyces cerevisiae. tRNA from the dimorphic fungus Candida albicans was found to have significant UGA and UAG readthrough activity and this activity was present in tRNA extracted from both the yeast and the hyphal phase of the fungus. Unusually the efficiency of readthrough activity in vitro was not affected by the [psi] determinant. C. albicans tRNA was fractionated by one-dimensional and two-dimensional gel electrophoresis and both readthrough activities appeared to be associated with a single species of tRNA.