tRNA chemical methylation. In vitro and in vivo formation of 1,7-dimethylguanosine at high concentrations of methylating agents

The methylation patterns produced in Escherichia coli B tRNA by a range of concentrations of the weak carcinogen dimethyl sulphate were examined with the following results: 1. 1,7-Dimethylguanosine was found to be formed in high amounts in the tRNA methylation reaction at high concentrations of methylating agent. 2. The dialkylated compound was recovered mainly in the form of derivatives, the spectral and chromatographic behaviour of which varied according to the procedures used for their isolation. Similar results were obtained for the in vivo methylation of rat-liver tRNA: after administration of a very high dose of the powerful carcinogen dimethylnitrosamine, 1,7-dimethylguanosine was found in rat-liver tRNA. Moreover, the analysis of the time-course of nucleic acid methylation indicated that this dialkylated product was still present in rat-liver tRNA when the major product of alkylation, 7-methylguanine, had almost completely disappeared.     

Ammonium ions prevent methylation of uridine to ribothymidine inAzotobacter vinelandii tRNA

It was shown that tRNA fromAzotobacter vinelandii grown in the presence of ammonium chloride lacks ribothymidine while that grown in the absence of the ammonium salt contains this modified nucleoside. [³²P]-Labelled tRNA from this organism grown in a medium containing the ammonium salt was digested with RNase T1 and the pseudouridinecontaining tetranucleotide, common to all tRNAs was isolated and analysed for the nucleoside replacing the ribothymidine. It was found to be uridine. Cells previously labelled with [³²P]-phosphate in the ammonium salt medium were washed and incubated in the ammonium saltfree medium to test whether ribothymidine would be formed upon removal of the ammonium ions. Methylation of the uridine did not take place.     

1,7-Diethylguanosine formation in tRNA chemical ethylation by ethionine and ethylnitrosourea

The mechanism of ethionine carcinogenesis and more generally the relationship between alkylation of nucleic acids by chemical carcinogens and oncogenesis still remain obscure. In the present study the rat liver tRNA ethylation by L-[ethyl-1-3H]ethionine was reinvestigated by examining in particular the highly radioactive 'pyrimidine-nucleotide-like' fraction found earlier in acid hydrolysates of hepatic tRNA from ethionine-treated rats. The following results were obtained: (1) ultraviolet-spectral and chromatographic analyses showed the presence of 1,7-diethylguanosine in this 'pyrimidine-nucleotide-like' fraction; (2) the dialkyl compound was recovered exclusively in the form of imidazole-ring-opened derivatives. When [1-14C]ethylnitrosourea was used as alkylating agent, the in vivo ethylation pattern of tRNA from various organs of rat showed an analogous radioactive 'pyrimidine-nucleotide-like' fraction as main radioactive product. On the contrary, tRNA ethylation pattern after in vitro reaction with [1-14C]ethylnitrosourea exhibited a main radioactivity peak (85% of the total radioactivity recovered) in coincidence of the chromatographic area of 1,7-diethylguanine. The 1,7-diethylguanosine moieties of tRNA were extremely labile both under physiological and alkaline conditions. The 1,7-diethylguanine-associated radioactivity was completely lost from [14C]ethyl-tRNA after only 7 h incubation at 37 degrees C and pH 7.3, while at pH 11.4 this process was preceded by the conversion of the 1,7-diethylguanosine residues into imidazole-ring-opened derivatives.     

Identity elements required for enzymatic formation of N2,N2-dimethylguanosine from N2-monomethylated derivative and its possible role in avoiding alternative conformations in archaeal tRNA

Here, we have investigated the specificity of purified recombinant tRNA:m(2)(2)G10 methyltransferase of Pyrococcus abyssi ((Pab)Trm-m(2)(2)G10 enzyme). This archaeal enzyme catalyses mono- and dimethylation of the N(2)-exocyclic amino group of guanine at position 10 of several tRNA species. Our results indicate that only few identity elements are required for the efficient formation of m(2)(2)G10. They are composed of a G10.U25 wobble base-pair in the dihydrouridine arm (D-arm) and a four nucleotide variable loop (V-loop) within a canonical three-dimensional (3D) structure. The types of base-pairs in the D-arm or amino acid acceptor stem are also important for the enzymatic reaction, but appear to affect only the rate of tRNA methylation. However, in tRNA species harbouring a G10-C25 Watson-Crick base-pair and/or five nucleotide V-loop, only m(2)G10 is produced. To impair the monomethylation reaction, drastic amputation in the T-arm is required. Our observations contrast with those reported earlier for the identity elements required for a remotely related Pyrococcus furiosus Trm-m(2)(2)G26 enzyme (alias (Pfu)Trm1) that also catalyses the two step formation of m(2)(2)G but at position 26 in several tRNA species. In this case, a G10-C25 base-pair together with the five nucleotide V-loop were shown to be required for efficient formation of m(2)(2)G26. Thus, in the Pyrococcus genus, the major identity elements that preclude formation of m(2)(2)G at positions 10 or 26 in tRNA are mutually exclusive. Therefore, the Trm-m(2)(2)G10 and Trm-m(2)(2)G26 enzymes have evolved independently towards different specificities. In addition, identity elements for m(2)/m(2)(2)G10 formation in archaeal tRNA are different from the ones required for m(2)G10 formation in eukaryal tRNA. We propose that archaeal tRNA:m(2)(2)G10 methyltransferases, unlike the orthologous eukaryal tRNA:m(2)G10 methyltransferases, evolved towards m(2)(2)G10 specificity due to the possible requirement of preventing formation of alternative structures in G/C rich archaeal tRNA species.     

Synthesis and function of isopentenyl adenosine derivatives in tRNA

Isopentenyl adenosine derivatives can be found next to the anticodon (position 37) in tRNA from both the Bacteria and Eucarya domains. These modified nucleosides improve the efficiency of tRNA in translation, can increase and decrease translational fidelity, and make the tRNA less codon context sensitive. In bacteria the synthesis of isopentenyl adenosine derivatives seems to be linked to iron metabolism and central metabolic pathways.     

Improved separation of modified nucleosides from tRNA hydrolysates: the patterns of tRNA methylation in rat tissues

A sensitive and reproducible method for the isolation of minor nucleosides derived from tRNA is described. The nucleosides obtained from enzymatic digestion of tRNA are separated into several groups using a QAE Sephadex column and increasing concentrations of boric acid in a step-wise manner. The nucleosides in each group are separated by isocratic elution from a preparative Partisil 10-SCX column and high-performance liquid chromatography at ambient temperature. With this method we have determined the patterns of tRNA methylation in vitro with extracts from rat bone, liver, kidney and adrenal glands. Although different tissues appear to contain the same tRNA methyltransferases, the patterns of methylated nucleosides are different.     

RNA Ligation and the Origin of tRNA

A straightforward origin of transfer RNA,(tRNA), is difficult to envision because of the apparentlycomplex idiosyncratic interaction between the D-loop and T-loop. Recently, multiple examples of the T-loop structuralmotif have been identified in ribosomal RNA. These examplesshow that the long-range interactions between the T-loop andD-loops seen in tRNA are not an essential part of the motifbut rather are facilitated by it. Thus, the core T-loopstructure could already have existed in a small RNA prior tothe emergence of the tRNA. The tRNA might then have arisenby expansion of an RNA that carried the motif. With thisidea in mind, Di Giulio's earlier hypothesis that tRNAevolved by a simple duplication or ligation of a minihelixRNA was re-examined. It is shown that an essentially moderntRNA structure can in fact be generated by the ligation oftwo 38-nucleotide RNA minihelices of appropriate sequence.Although rare, such sequences occur with sufficientfrequency, (1 in 3 × 10⁷), that they could be found in astandard in vitro RNA selection experiment. Theresults demonstrate that a series of RNA duplications, aspreviously proposed, can in principal account for the originof tRNA. More generally, the results point out that RNAligation can be a powerful driving force for increasedcomplexity in the RNA World.     

Enzymatic mechanism of tRNA (mU54)methyltransferase

tRNA (m⁵U54)methyltransferase (RUMT) catalyzes the methylation of uridine 54 of transfer RNA by S-adenosyl-l-methionine. In this report, we present the enzymatic mechanism of RUMT, including the stereochemical course of the methylation reaction, and discuss RUMT-tRNA recognition. As part of its enzymatic mechanism, we postulate that RUMT catalyzes the disruption of RNA-RNA contacts. We also show that many nucleotide substitutions can be made in the T-loop of tRNA without affecting RUMT binding, indicating that the recognition of the T-loop by RUMT is not stringent.     

Distinct origins of tRNA(m1G37) methyltransferase

The enzyme tRNA(m1G37) methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the N1 position of G37 in the anticodon loop of a subset of tRNA. The modified guanosine is 3' to the anticodon and is important for maintenance of reading frame during decoding of genetic information. While the methyltransferase is well conserved in bacteria and is easily identified (encoded by the trmD gene), the identity of the enzyme in eukarya and archaea is less clear. Here, we report that the enzyme encoded by Mj0883 of Methanocaldococcus jannaschii is the archaeal counterpart of the bacterial TrmD. However, despite catalyzing the same reaction and displaying similar enzymatic properties, MJ0883 and bacterial TrmD are completely unrelated in sequence. The catalytic domain of MJ0883, when aligned with the five known structural folds (I-V) that have been described to bind AdoMet, is of the class I fold, similar to the ancient Rossmann fold that binds nucleotides. In contrast, the catalytic domain of the bacterial TrmD has the unusual class IV fold of a trefoil knot structure. Thus, both the sequence and structural arrangements of tRNA(m1G37) methyltransferase have distinct evolutionary origins among primary kingdoms, revealing an unexpected but remarkable non-orthologous gene displacement to achieve an important tRNA modification.     

Overexpression of initiator methionine tRNA leads to global reprogramming of tRNA expression and increased proliferation in human epithelial cells

Transfer RNAs (tRNAs) are typically considered housekeeping products with little regulatory function. However, several studies over the past 10 years have linked tRNA misregulation to cancer. We have previously reported that tRNA levels are significantly elevated in breast cancer and multiple myeloma cells. To further investigate the cellular and physiological effects of tRNA overexpression, we overexpressed tRNA_i^Met in two human breast epithelial cell lines. We then determined tRNA abundance changes and performed phenotypic characterization. Overexpression of tRNA_i^Met significantly altered the global tRNA expression profile and resulted in increased cell metabolic activity and cell proliferation. Our results extend the relevance of tRNA overexpression in human cells and underscore the complexity of cellular regulation of tRNA expression.     

DNA methyltransferase homologue TRDMT1 in Plasmodium falciparum specifically methylates endogenous aspartic acid tRNA

In eukaryotes, cytosine methylation regulates diverse biological processes such as gene expression, development and maintenance of genomic integrity. However, cytosine methylation and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, here we investigated the presence of cytosine methylation in the nucleic acids of the protozoan Plasmodium falciparum. Interestingly, P. falciparum has TRDMT1, a conserved homologue of DNA methyltransferase DNMT2. However, we found that TRDMT1 did not methylate DNA, in vitro. We demonstrate that TRDMT1 methylates cytosine in the endogenous aspartic acid tRNA of P. falciparum. Through RNA bisulfite sequencing, we mapped the position of 5-methyl cytosine in aspartic acid tRNA and found methylation only at C38 position. P. falciparum proteome has significantly higher aspartic acid content and a higher proportion of proteins with poly aspartic acid repeats than other apicomplexan pathogenic protozoans. Proteins with such repeats are functionally important, with significant roles in host-pathogen interactions. Therefore, TRDMT1 mediated C38 methylation of aspartic acid tRNA might play a critical role by translational regulation of important proteins and modulate the pathogenicity of the malarial parasite.     

Insights into catalysis by a knotted TrmD tRNA methyltransferase

The crystal structure of Escherichia coli tRNA (guanosine-1) methyltransferase (TrmD) complexed with S-adenosyl homocysteine (AdoHcy) has been determined at 2.5A resolution. TrmD, which methylates G37 of tRNAs containing the sequence G36pG37, is a homo-dimer. Each monomer consists of a C-terminal domain connected by a flexible linker to an N-terminal AdoMet-binding domain. The two bound AdoHcy moieties are buried at the bottom of deep clefts. The dimer structure appears integral to the formation of the catalytic center of the enzyme and this arrangement strongly suggests that the anticodon loop of tRNA fits into one of these clefts for methyl transfer to occur. In addition, adjacent hydrophobic sites in the cleft delineate a defined pocket, which may accommodate the GpG sequence during catalysis. The dimer contains two deep trefoil peptide knots and a peptide loop extending from each knot embraces the AdoHcy adenine ring. Mutational analyses demonstrate that the knot is important for AdoMet binding and catalytic activity, and that the C-terminal domain is not only required for tRNA binding but plays a functional role in catalytic activity.     

The Origin and Evolution of tRNA Inferred from Phylogenetic Analysis of Structure

The evolutionary history of the two structural and functional domains of tRNA is controversial but harbors the secrets of early translation and the genetic code. To explore the origin and evolution of tRNA, we reconstructed phylogenetic trees directly from molecular structure. Forty-two structural characters describing the geometry of 571 tRNAs and three statistical parameters describing thermodynamic and mechanical features of molecules quantitatively were used to derive phylogenetic trees of molecules and molecular substructures. Trees of molecules failed to group tRNA according to amino acid specificity and did not reveal the tripartite nature of life, probably due to loss of phylogenetic signal or because tRNA diversification predated organismal diversification. Trees of substructures derived from both structural and statistical characters support the origin of tRNA in the acceptor arm and the hypothesis that the top half domain composed of acceptor and pseudouridine (TΨC) arms is more ancient than the bottom half domain composed of dihydrouridine (DHU) and anticodon arms. This constitutes the cornerstone of the genomic tag hypothesis that postulates tRNAs were ancient telomeres in the RNA world. The trees of substructures suggest a model for the evolution of the major functional and structural components of tRNA. In this model, short RNA hairpins with stems homologous to the acceptor arm of present day tRNAs were extended with regions homologous to TΨC and anticodon arms. The DHU arm was then incorporated into the resulting three-stemmed structure to form a proto-cloverleaf structure. The variable region was the last structural addition to the molecular repertoire of evolving tRNA substructures. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evolution of tRNA recognition systems and tRNA gene sequences

The aminoacylation of tRNAs by the aminoacyl-tRNA synthetases recapitulates the genetic code by dictating the association between amino acids and tRNA anticodons. The sequences of tRNAs were analyzed to investigate the nature of primordial recognition systems and to make inferences about the evolution of tRNA gene sequences and the evolution of the genetic code. Evidence is presented that primordial synthetases recognized acceptor stem nucleotides prior to the establishment of the three major phylogenetic lineages. However, acceptor stem sequences probably did not achieve a level of sequence diversity sufficient to faithfully specify the anticodon assignments of all 20 amino acids. This putative bottleneck in the evolution of the genetic code may have been alleviated by the advent of anticodon recognition. A phylogenetic analysis of tRNA gene sequences from the deep Archaea revealed groups that are united by sequence motifs which are located within a region of the tRNA that is involved in determining its tertiary structure. An association between the third anticodon nucleotide (N36) and these sequence motifs suggests that a tRNA-like structure existed close to the time that amino acid-anticodon assignments were being established. The sequence analysis also revealed that tRNA genes may evolve by anticodon mutations that recruit tRNAs from one isoaccepting group to another. Thus tRNA gene evolution may not always be monophyletic with respect to each isoaccepting group.Based on a presentation made at a workshop— Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code—held at Berkeley, CA, July 17–20, 1994 Correspondence to: M.E. Saks     

Classical molecular dynamics simulation of seryl tRNA synthetase and threonyl tRNA synthetase bound with tRNA and aminoacyl adenylate

Lacunae of understanding exist concerning the active site organization during the charging step of the aminoacylation reaction. We present here a molecular dynamics simulation study of the dynamics of the active site organization during charging step of subclass IIa dimeric SerRS from Thermus thermophilus (^ttSerRS) bound with ^tttRNA^Ser and dimeric ThrRS from Escherichia coli (^ecThrRS) bound with ^ectRNA^Thr. The interactions between the catalytically important loops and tRNA contribute to the change in dynamics of tRNA in free and bound states, respectively. These interactions help in the development of catalytically effective organization of the active site. The A76 end of the ^tttRNA^Ser exhibits fast dynamics in free State, which is significantly slowed down within the active site bound with adenylate. The loops change their conformation via multimodal dynamics (a slow diffusive mode of nanosecond time scale and fast librational mode of dynamics in picosecond time scale). The active site residues of the motif 2 loop approach the proximal bases of tRNA and adenylate by slow diffusive motion (in nanosecond time scale) and make conformational changes of the respective side chains via ultrafast librational motion to develop precise hydrogen bond geometry. Presence of bound Mg²⁺ ions around tRNA and dynamically slow bound water are other common features of both aaRSs. The presence of dynamically rigid Zinc ion coordination sphere and bipartite mode of recognition of ^ectRNA^Thr are observed.     

人线粒体tRNA基因突变与疾病

线粒体是体内重要的细胞器,存在于胞质内。许多生物化学反应都在线粒体内进行,其中尤为重要的是通过氧化磷酸化作用产生ＡＴＰ,供生命活动需要。线粒体内ｔＲＮＡ的总数只有２２个（核编码的ｔＲＮＡ总数至少为３０多个）。这样,线粒体ｔＲＮＡ与编码氨基酸之比基本是...     

镁离子稳定tRNA高级结构及其重要意义--生化所tRNA研究的一段历史回顾

镁离子稳定tRNA高级结构并具有重要的生理功能已经写入生物化学教科书。上世纪60年代初,中国科学院上海生物化学研究所的研究者与国外同行同时发现了这一现象。镁离子稳定tRNA的高级结构在tRNA的序列分析中可以得到较大的片段,甚至tRNA半分子。将这一事实运用于“片段重叠”法测定tRNA序列工作中,极大地推动了tRNA的序列测定。