Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “tRNA modification”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 937 records · Page 52Linked to original sources
PubMed2026

Quantitative RNA modification mapping by mass spectrometry with isobaric tags and nucleobase fragment analysis.

RNA modifications regulate RNA stability, translation, stress responses, and disease processes, yet their function remains poorly understood due to technical limitations in sequence analysis. Here, we present an RNA-specific isobaric tandem mass tagging (RMT) platform for omic-scale quantitative mapping of RNA modifications. The platform combines RNA-specific tags adapted from proteomics with an end-to-end workflow spanning sample preparation through data processing. Validation using synthetic oligonucleotides and total tRNA from Pseudomonas aeruginosa yielded reproducible quantification, with coefficients of variation below 5%. Together with nucleobase fragment analysis, we identified and quantified 24 RNA modifications in PA14 tRNAs, including previously undescribed m2A38 and Gm/Cm39, and assigned their corresponding writer enzymes. Further analyses of tRNAs from writer knockout strains and stressed cells revealed dynamic modification patterns, modification interdependencies, and their potential roles in stress adaptation. This method provides a robust, cost-effective platform for quantitative RNA modification mapping, enabling deeper biological insights.

RNA, Transfer↗
PubMed2003

The synthesis of oligoribonucleotides containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines via post-synthetic modification of precursor oligomers.

The N6-alkyladenosines and 2-methylthio-N6-alkyladenosines are the most common modified adenosine nucleosides and transfer ribonucleic acids (tRNA) are particularly rich in these modified nucleosides. They are present at position 37 of the anticodon arm and the contribution of these hypermodified nucleosides to codon-anticodon interactions, as well as translation, are significant, although not fully understood. Herein we described a new chemical synthesis method of the oligoribonucleotides containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines via post-synthetic modifications of precursor oligoribonucleotides. To obtain oligoribonucleotides containing N6-alkyladenosines, the precursor oligoribonucleotide carrying 6-methylthiopurine riboside residue was used, whereas for the synthesis of oligoribonucleotides containing 2-methylthio-N6-alkyladenosines the precursor oligoribonucleotide carrying the 2-methylthio-6-chloropurine riboside was applied. Among the modified oligoribonucleotides of different length and secondary structures, there were several containing naturally occurring modified nucleosides such as: N6-isopentenyladenosine (i6A), N6-methyladenosine (m6A), 2-methylthio-N6-isopentenyladenosine (ms2i6A), and 2-methylthio-N6-methyladenosine (ms2m6A), as well as several unnaturally modified adenosine derivatives.

Adenosine↗
PubMed1991

Site-specific incorporation of nonnatural residues during in vitro protein biosynthesis with semisynthetic aminoacyl-tRNAs.

A method is presented for the incorporation of nonnatural amino acids into proteins during in vitro cell-free translation. A combination of chemical synthesis and run-off transcription was employed to prepare a semisynthetic, nonhypermodified tRNA(Gly) nonsense suppressor acylated with L-3-[125I]iodotyrosine. The presence of this synthetic tRNA during in vitro translation of mRNA containing a nonsense suppression site (e.g., a UAG termination codon) results in the incorporation of the nonnatural amino acid L-3-iodotyrosine into the polypeptide exclusively at the position corresponding to that site. Incorporation of the nonnatural amino acid L-3-[125I]iodotyrosine into the model polypeptide was assessed by quantitative and unambiguous determination of suppression efficiency, read-through, and site specificity of incorporation. Minor modifications of the method employed in this initial experiment also allow the rapid analysis of unlabeled acylated tRNA analogues. Under optimum conditions, the unlabeled amino acid L-3-iodotyrosine was found to be incorporated with a suppression efficiency of 65%. Other nonnatural residues, including N-methylphenylalanine, D-phenylalanine, and phenyllactic acid, were tested in the assay under these same conditions. Suppression efficiencies for this series ranged from 0 to 72% depending on the structure of the residue incorporated. Several other aspects of this methodology, such as tRNA structure and context effects, are briefly discussed.

Acylation↗
PubMed1995

Characterization of a urea induced molten globule intermediate state of glutaminyl-tRNA synthetase from Escherichia coli.

The urea-induced unfolding of glutaminyl-tRNA synthetase, a multidomain protein, has been studied by equilibrium and kinetic methods, using chemical modification, fluorescence, and CD spectroscopy. The far-UV CD, fluorescence, and sulfhydryl reactivity clearly demonstrated the existence of a stable intermediate state at around 2 M urea. The intermediate showed higher binding of 1-anilino-8-naphthalenesulfonic acid. Furthermore, near-UV CD study of the intermediate showed significantly disrupted tertiary structure with only a small change in the secondary structure, which is a characteristic of molten globule states. The activation energies (delta G++) calculated from unfolding kinetics monitored by CD and fluorescence suggest that the intermediate state may be separated from the native and the unfolded state by high activation energy barriers.

Amino Acyl-tRNA Synthetases↗
PubMed1995

A novel approach to introduce site-directed specific cross-links within RNA-protein complexes. Application to the Escherichia coli threonyl-tRNA synthetase/translational operator complex.

We describe a methodology which allows the introduction of a photoactivatable azido group at specific internal positions of any RNA in order to identify the neighboring elements of an interacting protein. The first step involves site-directed modification of the target RNA with an antisense oligodeoxyribonucleotide bearing, at its 3' or 5' phosphate, a 4-[-N-(2-chloroethyl)-N-methylamino]benzylmethylamino group. Position N7 of a guanine residue located in the close vicinity of the hybrid is the main target for alkylation. The antisense oligodeoxyribonucleotide is then removed by acidic pH treatment and a photoreactive reagent (2,4-dinitro-5-fluorophenylazide) is condensed to the modified nucleotide. This method was used to induce specific cross-links between Escherichia coli threonyl-tRNA synthetase and the leader region of threonyl-tRNA synthetase mRNA, which is involved in translational feedback regulation. Control experiments revealed that the modification affects neither the structure of the mRNA nor the interaction with the enzyme. More than 50% of the modified mRNA complexed with threonyl-tRNA synthetase can be cross-linked to the enzyme, depending on the nucleotide modified.

Alkylation↗
PubMed1995

Transfer RNA aminoacylation: identification of a critical ribose 2'-hydroxyl-base interaction.

To understand the relationship between tRNA architecture and specific aminoacylation by aminoacyl-tRNA synthetases, we performed kinetic assays of Escherichia coli tRNA(Pro) molecules containing single deoxynucleotide substitutions. We identified an important 2'-hydroxyl group at position U8 (of 22 positions probed). Chemical modification studies showed that this 2'-hydroxyl interacts with either the N1 or the exocyclic amine of G46 in a hydrogen bonding interaction that contributes 1.8 kcal/mol to the free energy of activation for aminoacylation. Molecular modeling of tRNA(Pro) supports the existence of this interaction. This is the first study to identify a specific ribose 2'-hydroxyl-base interaction in the core region of a tRNA molecule that makes a thermodynamically significant contribution to aminoacylation.

Acylation↗
PubMed2006

Mechanistic insights into sulfur relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions.

The wobble bases of bacterial tRNAs responsible for NNR codons are modified to 5-methylaminomethyl-2-thiouridine (mnm5s2U). 2-thio modification of mnm5s2U is required for accurate decoding and essential for normal cell growth. We identified five genes yhhP, yheL, yheM, yheN, and yccK (named tusA, tusB, tusC, tusD, and tusE, respectively) that are essential for 2-thiouridylation of mnm5s2U by a systematic genome-wide screen ("ribonucleome analysis"). Efficient 2-thiouridine formation in vitro was reconstituted with recombinant TusA, a TusBCD complex, TusE, and previously identified IscS and MnmA. The desulfurase activity of IscS is stimulated by TusA binding. IscS transfers the persulfide sulfur to TusA. TusE binds TusBCD complex and stimulates sulfur transfer from TusA to TusD. TusE also interacts with an MnmA-tRNA complex. This study revealed that 2-thiouridine formation proceeds through a complex sulfur-relay system composed of multiple sulfur mediators that select and facilitate specific sulfur flow to 2-thiouridine from various pathways of sulfur trafficking.

Amino Acid Sequence↗
PubMed2004

Regulation of RNA function by aminoacylation and editing?

The chemical modification of nucleic acids is a ubiquitous phenomenon. Aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a reaction essentially devoted to protein synthesis but it is used also as an emergency mechanism to recycle stalled ribosomes, and it is required for genome replication in some RNA viruses. In several aminoacyl-tRNA synthetases a correction mechanism known as editing is present to prevent aminoacylation errors. Genome data reveal a growing number of open reading frames encoding ARS-like proteins. This strongly suggests the existence of a widespread and nonconventional machinery for aminoacylation and editing. Here we review the different biological functions of aminoacylation and editing; also we propose an evolutionary scenario for the origin of these two reactions, and hypothesize an extant role for RNA charging and editing outside the genetic code.

Amino Acyl-tRNA Synthetases↗
PubMed1992

Recognition of tertiary structure in tRNAs by Rh(phen)2phi3+, a new reagent for RNA structure-function mapping.

With photoactivation Rh(phen)2phi3+ promotes strand cleavage at sites of tertiary interaction in tRNA. The rhodium complex, which binds double-helical DNA by intercalation in the major groove, yields no cleavage in double-helical regions of the RNA or in unstructured single-stranded regions. Instead, Rh(phen)2phi3+ appears to target regions which are structured so that the major groove is open and accessible for stacking with the complex, as occurs where bases are triply bonded. So as to examine the specificity of this novel reagent and to evaluate its use in probing structural changes in RNAs, cleavage studies have been conducted on two structurally characterized tRNAs, tRNA(Phe) and tRNA(Asp) from yeast, the unmodified yeast tRNA(Phe) transcript, and a chemically modified tRNA(Phe), as well as on a series of tRNA(Phe) mutants. On tRNA(Phe) strong cleavage is observed at residues G22, G45, U47, psi 55, and U59; weaker cleavage is observed at A44, m7G46, and C48. On tRNA(Asp) cleavage is found at residues A21 through G26, psi 32, and U48, with minor cleavage apparent at A44, G45, A46, psi 55, U59, and U60. There is a striking similarity in cleavage observed on these tRNAs, and the sites of cleavage mark regions of tertiary folding. Cleavage on the unmodified tRNA(Phe) transcript resembles closely that found on native yeast tRNA(Phe), but additional sites, primarily in the anticodon loop and stem, are evident. The results indicate that globally the structures containing or lacking the modified bases appear to be the same; the differences in cleavage observed may reflect a loosening or alteration in the structure due to the absence of the modified bases. Cleavage results on mutants of tRNA(Phe) illustrate Rh(phen)2phi3+ as a sensitive probe in characterizing tRNA tertiary structure. Results are consistent with other assays for structural or functional changes. Uniquely, Rh(phen)2phi3+ appears to target directly sites of tertiary interaction. Cleavage results on mutants which involve base changes within the triply bounded region of the molecule indicate that it is the structure of the triply bonded array rather than the individual nucleotides which are being targeted. Chemical modification to promote selective depurination of the third base (m7G46) involved in the triple in the folded, native tRNA leads to the reduction of cleavage by the metal complex; this result shows directly the importance of the stacked triple base structure for recognition by the metal complex.(ABSTRACT TRUNCATED AT 400 WORDS)

2,2'-Dipyridyl↗
PubMed1989

Chloroplast-like transfer RNA genes expressed in wheat mitochondria.

In the course of a systematic survey of wheat mitochondrial tRNA genes, we have sequenced chloroplast-like serine (trnS-GGA), phenylalanine (trnF-GAA) and cysteine (trnC-GCA) tRNA genes and their flanking regions. These genes are remnants of 'promiscuous' chloroplast DNA that has been incorporated into wheat mtDNA in the course of its evolution. Each gene differs by one or a few nucleotides from the authentic chloroplast homolog previously characterized in wheat or other plants, and each could potentially encode a functional tRNA whose secondary structure shows no deviations from the generalized model. To determine whether these chloroplast-like tRNA genes are actually expressed, wheat mitochondrial tRNAs were resolved by a series of polyacrylamide gel electrophoreses, after being specifically end-labeled in vitro by 3'-CCA addition mediated by wheat tRNA nucleotidyltransferase. Subsequent direct RNA sequence analysis identified prominent tRNA species corresponding to the mitochondrial and not the chloroplast trnS, trnF and trnC genes. This analysis also revealed chloroplast-like elongator methionine, asparagine and tryptophan tRNAs. Our results suggest that at least some chloroplast-like tRNA genes in wheat mtDNA are transcribed, with transcripts undergoing processing, post-transcriptional modification and 3'-CCA addition, to produce mature tRNAs that may participate in mitochondrial protein synthesis.

Base Sequence↗
PubMed1991

O-ribosyl-phosphate purine as a constant modified nucleotide located at position 64 in cytoplasmic initiator tRNAs(Met) of yeasts.

The unknown modified nucleotide G*, isolated from both Schizosaccharomyces pombe and Torulopsis utilis initiator tRNAs(Met), has been identified as an O-ribosyl-(1"----2')-guanosine-5"-phosphate, called Gr(p), by means of HPLC, UV-absorption, mass spectrometry and periodate oxidation procedures. By comparison with the previously published structure of Ar(p) isolated from Saccharomyces cerevisiae initiator tRNA(Met), the (1"----2')-glycosidic bond in Gr(p) has been postulated to have a beta-spatial conformation. The modified nucleotide Gr(p) is located at position 64 in the tRNA(Met) molecules, i.e. at the same position as Ar(p). Since we have also characterized Gr(p) in Candida albicans initiator tRNA(Met), the phosphoribosylation of purine 64 can be considered as a constant nucleotide modification in the cytoplasmic initiator tRNAs(Met) of all yeast species so far sequenced. Precise evidence for the presence of Gr(p) in initiator tRNAs(Met) of several plants is also reported.

Candida↗
PubMed2003

Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli.

The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA(Ser)(CGA), an Arabidopsis intron-containing tRNA(Tyr)(GTA) and an Arabidopsis intron-containing tRNA(Met)(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of beta-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA(Ser) gene and the GUS reporter gene resulted in approximately 10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNA(Tyr) or tRNA(Met) genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA(Met)(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNA(Tyr) to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNA(Tyr) and tRNA(Met) were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.

Arabidopsis↗
PubMed1977

Change of protein reactivity in mammalian ribosomal subunits as a function of temperature.

Reductive methylation of rat liver ribosomal subunits was carried out at different temperatures and the reaction was followed as a function of time. The percentage of protein lysine residues which were methylated reached a definite plateau for each temperature, and was considerably increased by heating (four times at 40 degrees C). This increase was not observed when free ribosomal proteins were heated under the same conditions. Ribosomal subunits kept their biological activity (elongation steps of protein synthesis) even when methylated to a high extent. Half of the subunit activity was still found when 67% of the lysine residues were methylated. Proteins were divided into different classes on the basis of their alkylation in response to temperature. Those which reacted poorly within subunits even at high temperature should interact directly with rRNA. The results suggest that the protein reactivity modifications induced by heating (up to 37 degrees C), which were often totally or partially reversible, reflect a conformational change of the proteins themselves within the subunits. Besides protein unfolding, higher temperatures produced structural modification and inactivation of the subunits, which were restrained by the presence of aminoacyl tRNA.

Animals↗
PubMed1991

In vivo collagen turnover during development of thyroxine-induced left ventricular hypertrophy.

Cardiac fibroblasts synthesize large amounts of procollagens, yet only a small fraction of mature collagens accumulate in the extracellular matrix. To determine the roles of intracellular degradation of newly synthesized procollagens and extracellular degradation of mature collagens during normal growth and during thyroxine-induced left ventricular hypertrophy, in vivo left ventricular procollagen synthetic rates were assessed in control rats and rats treated with L-thyroxine for 1, 2, 4, and 8 wk (1 mg.kg-1.day-1). A modification of the flooding infusion method was developed using measurements of cardiac prolyl-tRNA, and tissue-free and protein-bound hydroxyproline specific radioactivities 60 min after intravenous administration of a massive dose of [3H]proline. Degradative rates of newly synthesized procollagens and mature collagens were then derived as the difference between rates of procollagen synthesis and collagen accumulation. Left ventricular procollagen synthetic rates were markedly increased after 1 wk of hormone administration (256 +/- 16 and 166 +/- 13 micrograms/day per left ventricle for thyroxine-treated and control animals, respectively; P less than 0.01). An even greater increase in procollagen synthetic rates was observed after 8 wk (438 +/- 46 and 202 +/- 18 micrograms/day for thyroxine-treated and control animals, respectively; P less than 0.01). Despite increased procollagen synthesis, disproportionate accumulation of fibrillar collagens (assessed as the relative concentration of protein-bound hydroxyproline in left ventricular tissue) did not occur. Derived left ventricular degradative rates for newly synthesized procollagens as well as for mature collagens were increased in thyroxine-treated animals. Increased procollagen synthesis, enhanced flux of newly synthesized procollagens through intracellular degradative pathways, and extensive extracellular matrix remodeling without disproportionate collagen accumulation are characteristics of this form of "physiological" left ventricular hypertrophy.

Animals↗
PubMed1992

RNA metabolism during the sexual differentiation of rat hypothalamus.

Modifications in the basal molecular biology parameters (total concentrations of RNA, DNA, proteins, rRNA, tRNa, free and polysomal bound poly-A+ mRNA) have been determined daily in the growing hypothalamus of male and female rats from day 1 to day 8 after birth. Changes observed in the parameters studied in this work occurred mainly in the first 48 h after birth. In males tRNA and free mRNA (f-mRNA) contents decreased from day 1 to day 2 and then their concentrations remained more or less constant. Total mRNA significantly decreased from day 1 to day 3 and showed a further significant decrease from day 6 to day 8. Polysomal bound-mRNA (b-mRNA) decreased from day 1 to day 3, then increased to day 6, and finally decreased once more from day 6 to day 8. The b-mRNA/f-mRNA, mRNA/rRNA and mRNA/total RNA ratios showed a bimodal behavior with a first peak on day 2, and a second, smaller peak, on days 6-7. The changes observed in the females on the first 2-3 days of life were the inverse of those observed in the males, most of the parameters studied showed a sharp increase from day 1 to day 2 or to day 3 and then a drastic decrease. The only exception to this behavior was the b-mRNA/f-mRNA ratio which showed a small decrease from day 1 to day 2, followed by a continuous increase from day 2 to day 8. b-mRNA concentrations, after the sharp decrease from day 2 to day 3 of life, increased from day 3 to day 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

[Binding of a fragment of yeast phenylalanyl tRNA containing an anticodon loop with 30S and 70S ribosomes from Escherichia coli. The role of guanosine-42 in this interaction].

Poly(U)-dependent binding of isolated yeast tRNA(Phe) anticodon hairpin (15-nucleotide-long, corresponding to nucleotides 28-42 within the tRNA) and several its derivatives to the P site of Escherichia coli 30S and 70S ribosomes was studied quantitatively. The affinity for the hairpin binding to 70S ribosomes was shown to be only 30-fold weaker than that for the binding of total tRNA(Phe). Within the anticodon hairpin, removal of the 3'-terminal nucleotide corresponding to guanosine-42 in tRNA(Phe) decreases the association constant for the anticodon arm-ribosome interaction 15-fold. Replacement of this guanosine with other nucleosides does not affect the affinity, regardless of involvement in the hairpin secondary structure. These data indicate that G-42 affects the anticodon arm affinity most likely by forming a direct contact with the ribosome. One can assume that this nucleotide within intact tRNA also forms a contact with the P site. Since the 3'-terminal ribose modifications (oxidation, oxidation and reduction) as well as the presence or absence of the 3'-terminal phosphate does not affect the affinity of the anticodon arm fragment, the latter is obviously involved in the interaction through 3'-terminal nucleotide base groups which does not take part in base pairing.

Anticodon↗
PubMed1999

Transfer RNA modification enzymes from Pyrococcus furiosus: detection of the enzymatic activities in vitro.

The modification patterns of in vitro transcripts of two yeast Saccharomyces cerevisiae tRNAs (tRNAPheand tRNAAsp) and one archaeal Haloferax volcanii tRNA (tRNAIle) were investigated in the cell-free extract of Pyrococcus furiosus supplemented with S -adenosyl-l-methionine (AdoMet). The results indicate that the enzymatic formation of 11 distinct modified nucleotides corresponding to 12 enzymatic activities can be detected in vitro. They correspond to the formation of pseudouridines (Psi) at positions 39 and 55, 2' -O- ribose methylations at positions 6 (Am) and 56 (Cm), base methylations at positions 10 (m2G), 26 (m22G), 37 (m1G), 49 (m5C), 54 (m5U) and 58 (m1A) and both the deamination and methylation of adenosine into m1I at position 57. Most of the detected modified nucleotides are common modifications found in other phylogenetic groups, while Am6, Cm56and m1I57are specific modifications found exclusively in Archaea. It is also shown that the enzymatic formation of m5C49, m5U54, Psi55and m1I57does not depend on the three-dimensional architecture of the tRNA substrate, since these modi-fications also occur in fragmented tRNAs as substrate.

Base Sequence↗
PubMed2002

Influence of the relA gene on ribosome frameshifting.

We have examined the influence of genotype at the relA locus on the kinetics of leftward (or -1) frameshifting at a variety of codons calling for a limiting aminoacyl-tRNA species. We used lacZ left-frameshift reporter constructs carrying the sequenceU UUC XYZ, whereXYZ was each of three triplets coding for three different amino acids; we slowed the ribosomes at each of these by limiting for the amino acid or for the aminoacyl-tRNA. In all cases, limitation stimulated leftward frameshifting. In all cases, the stimulation was greater in relA mutant cells than in their wild-type relA(+) counterparts. In the latter genotype, the increased frameshifting was constant from the start of the limitation regime. This was also true of the relA mutant strain during limitation for lysine-tRNA or for leucine; however, during limitation for isoleucine-tRNA (or for isoleucine) the mutant showed a gradual, progressive increase in frameshifting, suggesting an indirect effect. We suggest that gradual accumulation of undermodified tRNAs, which is characteristic of the relA response, is involved. However, the specific modification involved is unknown. It is not queosine: analysis of a tgt mutant that is completely defective in queosine modification showed no increase in leftward frameshifting on the reporter which showed the larger, gradual increase during the relA response to isoleucine-tRNA limitation.

Escherichia coli↗