tRNAPhe cleavage by aminoglycosides is triggered off by formation of an abasic site

This communication reports the characteristics of the mechanism of highly specific tRNA(Phe) cleavage, which occurs in the anticodon loop in the presence of aminoglycoside antibiotic-neomycin B. The data prove that the cleavage requires previous depurination of the polynucleotide chain at position 37, which is occupied by a hypermodified guanine base-wybutine. The results suggest that the phenomenon, previously considered as selective with respect to the presence of tRNA hypermodification, may concern far more RNA molecules, namely the ones carrying abasic sites.     

Structural changes of tRNA and 5S rRNA induced with magnesium and visualized with synchrotron mediated hydroxyl radical cleavage

The structure of native yeast tRNA^Phe and wheat germ ribosomal 5S RNA induced by different magnesium ion concentrations was studied in solution with a synchrotron mediated hydroxyl radical RNA cleavage reaction. We showed that very small amounts of Mg⁺² can induce significant changes in the hydroxyl radical cleavage pattern of tRNA^Phe. It also turned out that a reactivity of tRNA^Phe towards OH coincides with the strong metal binding sites. Because of the Mg ions are heavily hydrated one can suggest the strong correlation of the observed nucleosides reactivity in vicinity of Mg²⁺ binding sites with availability of water molecules as a source of hydroxyl radical. On the other hand the structure of wheat germ 5S rRNA is less sensitive to the hydroxyl radical reaction than tRNA^Phe although some changes are visible at 4 mM Mg ions. It is probably due to the lack of strong Mg⁺² binding sites in that molecule. The reactivity of nucleotides in loops C and D of 5S rRNA is not effected, what suggests their flexibility or involvement in higher order structure formation. There is different effect of magnesium on tRNA and 5S rRNA folding. We found that nucleotides forming strong binding sites for magnesium are very sensitive to X-ray generated hydroxyl radical and can be mapped with OH. The results show, that guanine nucleotides are preferentially hydrated. X-ray footprinting mediated hydroxyl radical RNA cleavage is a very powerful method and has been applied to studies of stable RNAs for the first time.     

The influence of spermine on the structural dynamics of yeast tRNA

A tRNA^Phe derivative carrying ethidium at position 37 in the anticodon loop has been used to study the effect of spermine on conformational transitions of the tRNA. As previously reported (Ehrenberg, M., Rigler, R. and Wintermeyer, W. (1979) Biochemistry 18, 4588–4599) in the tRNA derivative the ethidium is present in three states (T₁–T₃) characterized by different fluorescence decay rates. T-jump experiments show two transitions between the states, a fast one (relaxation time 10–100 ms) between T₁ and T₂, and a slow one (100–1000 ms) between T₂ and T₃. In the presence of spermine the fast transition shows a negative temperature coefficient indicating the existence of a preequilibrium with a negative reaction enthalpy. Spermine shifts the distribution of states towards T₃, as does Mg²⁺, but the final ratio obtained with spermine is higher than with Mg²⁺, which we tentatively interpret to mean that spermine stabilizes one particular conformation of the anticodon loop.     

Human DNMT2 methylates tRNA(Asp) molecules using a DNA methyltransferase-like catalytic mechanism

Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNA(Asp). We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNA(Asp) confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism.     

The Seryl-tRNA synthetase/tRNA acceptor stem interface is mediated via a specific network of water molecules

tRNAs are aminoacylated by the aminoacyl-tRNA synthetases. There are at least 20 natural amino acids, but due to the redundancy of the genetic code, 64 codons on the mRNA. Therefore, there exist tRNA isoacceptors that are aminoacylated with the same amino acid, but differ in their sequence and in the anticodon. tRNA identity elements, which are sequence or structure motifs, assure the amino acid specificity. The Seryl-tRNA synthetase is an enzyme that depends on rather few and simple identity elements in tRNA^Ser. The Seryl-tRNA-synthetase interacts with the tRNA^Ser acceptor stem, which makes this part of the tRNA a valuable structural element for investigating motifs of the protein–RNA complex. We solved the high resolution crystal structures of two tRNA^Ser acceptor stem microhelices and investigated their interaction with the Seryl-tRNA-synthetase by superposition experiments. The results presented here show that the amino acid side chains Ser151 and Ser156 of the synthetase are interacting in a very similar way with the RNA backbone of the microhelix and that the involved water molecules have almost identical positions within the tRNA/synthetase interface.     

Effect of modification of tRNA nucleotide 37 on the tRNA interaction with the A and P sites of the Escherichia coli 70S ribosome

The modified nucleotide 3′ of the tRNA anticodon is an important structural element that regulates the codon-anticodon interaction in the ribosome by stacking with codon-anticodon bases. The presence and identity (pyrimidine, purine, or modified purine) of this nucleotide significantly affects the energy of stacking in the A and P sites of the ribosome. Modification of nucleotide 37 does not contribute to stacking in the A site of the 70S ribosome, while its effect is substantial in the P site. The enthalpies of tRNA interactions with the A and P sites in the ribosome are similar and considerably lower than the enthalpy of the interactions of two tRNAs with the cognate anticodons in solution, suggesting that the ribosome contributes to the enthalpy-related portion of the free energy of tRNA binding by directly forming additional interactions with tRNA or by indirectly stabilizing the conformation of the codon-anticodon complex. In addition to stacking, tRNA binding in the A and P sites is further stabilized by interactions that involve magnesium ions. The number of ions involved in the formation of the tRNA-ribosome complex depends on the identity of tRNA nucleotide 37.     

Unlike in other monocotyledonous plants such as wheat and rice, the region containing the tRNA (UCC), tRNA (UCU) and CF1 ATPase α-subunit genes has not undergone recombination in the leek chloroplast DNA

The nucleotide sequence of a 1.1 kbp BamHI fragment of the leek chloroplast DNA (Allium porrum., fam. Liliaceae) has been determined. The fragment contains the 3' part of the tRNA^Gly (UCC) gene and the tRNA^Arg (UCU) gene on the same strand, and the 3' end of the atpA gene encoding the CF₁ ATPase α-subunit which is located on the opposite strand. The gene arrangement and nucleotide sequence of this fragment are similar to those of the corresponding region in the tobacco chloroplast DNA but differ significantly from what has been observed in other monocotyledonous plants such as wheat and rice, in which the region containing these genes has undergone intensive rearrangement.     

Domains of phenylalanyl-tRNA synthetase from Thermus thermophilus required for aminoacylation

The contribution of entire domains or particular amino acid residues of the phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus to the interaction with tRNA^Phe was studied. Removal of domain 8 of the β subunit resulted in drastic reduction of the dissociation constant of the FRS·tRNA^Phe complex. Neither the removal of arginine 2 of the β subunit, which makes the only major contact between domains β1–5 and the tRNA, nor the replacement of the conserved proline 473 by glycine had an influence on the aminoacylation activity of the FRS. Thus, the body comprising domains 1–5 of the β subunit may not be essential for efficient aminoacylation of tRNA^Phe by the FRS and rather be involved in other functions.     

Rodent type 2 Alu family,rat identifier sequence,rabbit C family,and bovine or goat 73-bp repeat may have evolved from tRNA genes

Summary Close structural resemblances between several mammalian highly or moderately repetitive families and some specific tRNAs were detected. The rodent type 2 Alu family, rat identifier (ID) sequences, rabbit C family, and bovine or goat 73-bp repeat are most homologous with lysine tRNA₅, phenylalanine tRNA, glycine tRNA, and glycine tRNA, respectively. The homologies extend to secondary structures, and the homologous nucleotides are located on nearly the same secondary structures. The repetitive families mentioned have a common structural organization, with a tRNA-like sequence devoid of an aminoacyl stem region. These features suggest that these repetitive families may be generated by nonhomologous recombination between a tRNA gene and a tRNA-unrelated block.     

Crystal structure of the E. coli tRNA aminoacyl stem isoacceptor RR-1660 at 2.0 Å resolution

Due to the redundancy of the genetic code there exist six mRNA codons for arginine and several tRNA^Arg isoacceptors which translate these triplets to protein within the context of the mRNA. The tRNA identity elements assure the correct aminoacylation of the tRNA with the cognate amino acid by the aminoacyl-tRNA-synthetases. In tRNA^Arg, the identity elements consist of the anticodon, parts of the D-loop and the discriminator base. The minor groove of the acceptor stem interacts with the arginyl-tRNA-synthetase. We crystallized different Escherichia coli tRNA^Arg acceptor stem helices and solved the structure of the tRNA^Arg isoacceptor RR-1660 microhelix by X-ray structure analysis. The acceptor stem helix crystallizes in the space group P1 with the cell constants a = 26.28, b = 28.92, c = 29.00 Å, α = 105.74, β = 99.01, γ = 97.44° and two molecules per asymmetric unit. The RNA hydration pattern consists of 88 bound water molecules. Additionally, one glycerol molecule is bound within the interface of the two RNA molecules.     

Effect of mitochondrial tRNA mutation on the clinical and biochemical characteristics of Chinese essential hypertensive subjects

Mitochondrial dysfunction has been potentially implicated in both human and experimental hypertension. We performed the mutational analysis of tRNA^Lys gene by PCR amplification and subsequent sequence analysis of the PCR fragments from 990 Chinese essential hypertensive subjects. We also made a comparative analysis of the collected data of the essential hypertension subjects who carried tRNA^Lys mutation and those who did not carry the mutation using the methods of 1:1 case-control study. We totally found 7 mutation sites in 10 subjects. The onset ages of the individuals carrying the mutation were earlier than those who did not bear them. The level of blood urea nitrogen in hypertension subjects who carried tRNA^Lys mutation was higher than the hypertension subjects who did not carried tRNA^Lys mutation, while the serum potassium was significantly lower. The level of platelet count in hypertension subjects who carried tRNA^Lys mutation was lower. The level of ventricular septal thickness in hypertension subjects who carried tRNA^Lys mutation was higher and the level of left ventricular end diastolic diameter in hypertension subjects was significantly lower. Mitochondrial tRNA^Lys mutations might result in the change of their structure and function, and then damaged the blood metabolism, the balance of the blood electrolyte, the steady-state of the blood cells and the heart structure and function, which were involved in the progress of the essential hypertension. Part of the essential hypertension patients clinically presented the characters of maternal inheritance, which might be associated with the tRNA^Lys mutation.     

The 1.2 Å crystal structure of an E. coli tRNA acceptor stem microhelix reveals two magnesium binding sites

tRNA identity elements assure the correct aminoacylation of tRNAs by the cognate aminoacyl-tRNA synthetases. tRNA^Ser belongs to the so-called class II system, in which the identity elements are rather simple and are mostly located in the acceptor stem region, in contrast to ‘class I’, where tRNA determinants are more complex and are located within different regions of the tRNA.The structure of an Escherichia coli tRNA^Ser acceptor stem microhelix was solved by high resolution X-ray structure analysis. The RNA crystallizes in the space group C2, with one molecule per asymmetric unit and with the cell constants a = 35.79, b = 39.13, c = 31.37 Å, and β = 111.1°. A defined hydration pattern of 97 water molecules surrounds the tRNA^Ser acceptor stem microhelix. Additionally, two magnesium binding sites were detected in the tRNA^Ser aminoacyl stem.     

Sequence analysis of three tRNAPhe nuclear genes and a mutated gene,and one gene for tRNAAla from Arabidopsis thaliana

Three genes and one mutant gene for tRNA^Phe (GAA) and one gene for tRNA^Ala (UGC) were isolated from a whole-cell DNA library of Arabidopsis thaliana. All three tRNA^Phe genes are identical in their nucleotide sequence, but differ in their 5 and 3 flanking regions. The mutant tRNA^Phe (GAA) gene differs from the other three genes by one nucleotide change from highly conserved G to C at the 57th nucleotide position. The primary structure of the first tRNA^Ala gene was also determined in this experiment.     

Superposition of two tRNA acceptor stem crystal structures: Comparison of structure, ligands and hydration

We solved the X-ray structures of two Escherichia coli tRNA^Ser acceptor stem microhelices. As both tRNAs are aminoacylated by the same seryl-tRNA-synthetase, we performed a comparative structure analysis of both duplexes to investigate the helical conformation, the hydration patterns and magnesium binding sites. It is well accepted, that the hydration of RNA plays an important role in RNA-protein interactions and that the extensive solvent content of the minor groove has a special function in RNA. The detailed comparison of both tRNA^Ser microhelices provides insights into the structural arrangement of the isoacceptor tRNA aminoacyl stems with respect to the surrounding water molecules and may eventually help us to understand their biological function at atomic resolution.     

Comparative X-ray structure analysis of human and Escherichia coli tRNA(Gly) acceptor stem microhelices

tRNA identity elements assure the correct aminoacylation of tRNAs by the aminoacyl-tRNA synthetases with the cognate amino acid. The tRNA^Gly/glycyl-tRNA sythetase system is member of the so-called ‘class II system’ in which the tRNA determinants consist of rather simple elements. These are mostly located in the tRNA acceptor stem and in the glycine case additionally the discriminator base at position 73 is required. Within the glycine-tRNA synthetases, the archaebacterial/human and the eubacterial sytems differ with respect to their protein structures and the required tRNA identity elements, suggesting a unique evolutionary divergence.In this study, we present a comparison between the crystal structures of the eubacterial Escherichia coli and the human tRNA^Gly acceptor stem microhelices and their surrounding hydration patterns.     

Crystal structure of the human tRNA microhelix isoacceptor G9990 at 1.18 Å resolution

The tRNA^Gly/Glycyl-tRNA synthetase system belongs to the so called ‘class II’ in which tRNA identity elements consist of relative few and simple motifs, as compared to ‘class I’ where the tRNA determinants are more complicated and spread over different parts of the tRNA, mostly including the anticodon. The determinants from ‘class II’ although, are located in the aminoacyl stem and sometimes include the discriminator base. There exist predominant structure differences for the Glycyl-tRNA-synthetases and for the tRNA^Gly identity elements comparing eucaryotic/archaebacterial and eubacterial systems.We focus on comparative X-ray structure analysis of tRNA^Gly acceptor stem microhelices from different organisms. Here, we report the X-ray structure of the human tRNA^Gly microhelix isoacceptor G9990 at 1.18 Å resolution. Superposition experiments to another human tRNA^Gly microhelix and a detailed comparison of the RNA hydration patterns show a great number of water molecules with identical positions in both RNAs. This is the first structure comparison of hydration layers from two isoacceptor tRNA microhelices with a naturally occurring base pair exchange.     

The crystal structure of a Thermus thermophilus tRNA acceptor stem microhelix at 1.6 Å resolution

tRNAs are aminoacylated with the correct amino acid by the cognate aminoacyl-tRNA synthetase. The tRNA/synthetase systems can be divided into two classes: class I and class II. Within class I, the tRNA identity elements that enable the specificity consist of complex sequence and structure motifs, whereas in class II the identity elements are assured by few and simple determinants, which are mostly located in the tRNA acceptor stem.The tRNA^Gly/glycyl-tRNA-synthetase (GlyRS) system is a special case regarding evolutionary aspects. There exist two different types of GlyRS, namely an archaebacterial/human type and an eubacterial type, reflecting the evolutionary divergence within this system. We previously reported the crystal structures of an Escherichia coli and of a human tRNA^Gly acceptor stem microhelix. Here we present the crystal structure of a thermophilic tRNA^Gly aminoacyl stem from Thermus thermophilus at 1.6 Å resolution and provide insight into the RNA geometry and hydration.     

Crystal structure of a human tRNA(Gly) microhelix at 1.2A resolution

The tRNA^Gly/glycyl-tRNA synthetase (GlyRS) system belongs to the so-called ‘class II aminoacyl-tRNA synthetase system’ in which tRNA identity elements are assured by rather few and simple determinants mostly located in the tRNA acceptor stem. Regarding evolutionary aspects, the tRNA^Gly/GlyRS system is a special case. There exist two different types of GlyRS, namely an archaebacterial/human type and a eubacterial type reflecting an evolutionary divergence within this system.Here we report the crystal structure of a human tRNA^Gly acceptor stem microhelix at 1.2 Å resolution. The local geometric parameters of the microhelix and the water network surrounding the RNA are presented. The structure complements the previously published Escherichia coli tRNA^Gly aminoacyl stem structure.