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 397 records · Page 22Linked to original sources
PubMed2002

Identification of Aquifex aeolicus tRNA (m2(2G26) methyltransferase gene.

The modifications of N2,N2-dimethylguanine (m2(2)G) are found in tRNAs and rRNAs from eukarya and archaea. In tRNAs, modification at position G26 is generated by tRNA (m2(2)G26) methyltransferase, which is encoded by the corresponding gene, trm1. This enzyme catalyzes the methyl-transfer from S-adenosyl-L-methionine to the semi-conserved residue, G26, via the intermediate modified base, m2G26. Recent genome sequencing project has been reported that the putative trm1 is encoded in the genome of Aquifex aeolicus, a hyper-thermophilic eubacterium as only one exception among eubacteria. In order to confirm whether this bacterial trm1 gene product is a real tRNA (m2(2)G26) methyltransferase or not, we expressed this protein by wheat germ in vitro cell-free translation system. Our biochemical analysis clearly showed that this gene product possessed tRNA (m2(2)G26) methyltransferase activity.

Bacteria↗
PubMed1979

Conformation transitions of a tRNA--aminoacyl-tRNA synthetase complex induced by tRNAs bearing different modifications in the 3' terminus.

The influence of modifications of the 3'-terminal adenosine of tRNAPhe (yeast) on the complex formation between this tRNA and phenylalanyl-tRNA synthetase (yeast) has been investigated by using fluorescence titrations and fast kinetic techniques. Subtle changes in the 3' terminus are reflected by distinct alterations in the two-step recognition process which had been demonstrated earlier for the native substrate tRNAPheCCA [Krauss, G., Riesner, D., & Maass, G. (1977) Nucleic Acids Res. 4, 2253--2262]. Binding experiments with tRNAPheCC, tRNAPheCCA-ox-red, tRNAPheCC2'dA, tRNAPheCC3'dA, tRNAPheCC-formycin, and tRNAPheCC-formycin-ox-red confirm that the 3'-terminal adenosine participates in a conformational change of the tRNA--synthetase complex. This is valid in both the absence and presence of phenylalaninyl-5'-AMP, the alkyl analogue of the aminoacyladenylate. As compared to tRNAPheCCA, a slower conformational change is observed with the competitive inhibitor tRNAPheCC-formycin-ox-red. The reaction enthalpy and/or the quench of the Y-base fluorescence that accompany the conformational change are altered upon binding of tRNAPheC2'dA, tRNAPheCC3'dA, and tRNAPheCC-formycin. It is evident that the final adaptation between tRNA and its synthetase in the complex is determined by the chemical nature of the 3'-terminal nucleotide. This is of vital importance for the specificity of the aminoacylation process.

Amino Acyl-tRNA Synthetases↗
PubMed1984

High resolution localization of the tRNA anticodon interaction site on the Escherichia coli 30 S ribosomal subunit.

A body of previous work has shown that when Escherichia coli tRNAVal1 is placed in the P site of E. coli ribosomes and irradiated, the 5'-anticodon base of this tRNA, 5-carboxymethoxyuridine, is cross-linked to C-1400 of the 16 S rRNA. By tagging the carboxyl group of the cross-linked tRNA residue with a 2,4-dinitrophenyl (DNP) group attached via a 9 A spacer, it has been possible to directly visualize this cross-linking site by immunoelectron microscopy. The DNP group was attached by addition of ethylenediamine to the carboxyl group, followed by condensation of the newly formed free amino group with the N-hydroxysuccinimide ester of N-2,4-dinitrophenyl-gamma-aminobutyric acid. When reacted with anti-DNP antibody, this modification brings the surface of the antibody to within 9 A of the pyrimidine ring which was cross-linked. Neither codon-dependent binding nor cross-linking were materially affected by the tRNA modification. The tRNA-ribosome adduct formed a stable complex with anti-DNP antibody only when 50-30 S subunit association was prevented. Electron microscopic examination of the immune complexes showed that greater than 95% of those detected had the antibody localized deep in the cleft which separates the head and neck of the 30 S from the large protrusion. Since this is the site of cross-linking of the anticodon of tRNA, we conclude that this region on the 30 S subunit corresponds to the decoding site.

Antibodies↗
PubMed1992

A magnesium-induced conformational transition in the loop of a DNA analog of the yeast tRNA(Phe) anticodon is dependent on RNA-like modifications of the bases of the stem.

Two single-stranded DNA heptadecamers corresponding to the yeast tRNA(Phe) anticodon stem-loop were synthesized, and the solution structures of the oligonucleotides, d(CCAGACTGAAGATCTGG) and d(CCAGACTGAAGAU-m5C-UGG), were investigated using spectroscopic methods. The second, or modified, base sequence differs from that of DNA by RNA-like modifications at three positions; dT residues were replaced at positions 13 and 15 with dU, and the dC at position 14 with d(m5C), corresponding to positions where these nucleosides occur in tRNA(Phe). Both oligonucleotides form intramolecular structures at pH 7 in the absence of Mg2+ and undergo monophasic thermal denaturation transitions (Tm = 47 degrees C). However, in the presence of 10 mM Mg2+, the modified DNa adopted a structure that exhibited a biphasic "melting" transition (Tm values of 23 and 52 degrees C) whereas the unmodified DNA structure exhibited a monophasic denaturation (Tm = 52 degrees C). The low-temperature, Mg(2+)-dependent structural transition of the modified DNA was also detected using circular dichroism (CD) spectroscopy. No such transition was exhibited by the unmodified DNA. This transition, unique to the modified DNA, was dependent on divalent cations and occurred most efficiently with Mg2+; however, Ca2+ also stabilized the alternative conformation at low temperature. NMR studies showed that the predominant structure of the modified DNA in sodium phosphate (pH 7) buffer in the absence of Mg2+ was a hairpin containing a 7-nucleotide loop and a stem composed of 3 stable base pairs. In the Mg(2+)-stabilized conformation, the loop became a two-base turn due to the formation of two additional base pairs across the loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

[Role of the carboxylic groups in interaction of phenylalanyl-tRNA synthetase with substrates].

The effect of modification of carboxylic groups of phenylalanyl-tRNA synthetase by p-toluene sulfonate N-cyclohexyl-N'-beta-(4-methylmorpholine) ethylcarbodiimide (CMEC) on the activity of the enzyme was investigated. It was shown that modification of two moles of carboxylic groups per mole of the enzymes leads to the diminution of negative charge of the enzyme and to inactivation in ATP-[32P]PPi-exchange and aminoacylation reactions. The inactivation is completely reversed by mild alkaline hydrolysis. ATP in concentration 2 X 10(-4) M partially protects the enzyme against inactivation, protective effect being stimulated by Mg2+ and 0.4-0.7 moles of carboxylic groups per mole of the enzyme are protected against inactivation is observed although the depth of modification is increased. Other substrates do not have protective effect. Modification of the enzyme by CMEC increases Kdiss value of [14C]-Phe-tRNA enzyme complex and Km value for tRNAPhe in aminoacylation by factor of three. Vmax for all substrates in both aminoacylation and leads to 40% increase of Hill's coefficient for ATP in ATP-[32P]PPi-exchange reaction but not in aminoacylation. The carboxylic groups modified by CMEC are assumed to take part in ATP recognition and in catalysis of the ATP conversion and in catalysis of transfer of activated amino acid residues on tRNA.

Adenosine Triphosphate↗
PubMed1980

tRNA binding stabilizes rat liver 60 S ribosomal subunits during treatment with LiCl.

We have shown recently that, in the absence of mRNA, 1 molecule of nonacylated tRNA binds to the large ribosomal subunit of rat liver with a high affinity constant (Buisson, M., Reboud, A.M., Dubost, S., and Reboud, J. P. (1979) Biochem. Biophys. Res. Commun. 90,634-640). In this paper, free and tRNA-bound 60 S subunits were treated with increasing concentrations of LiCl to obtain information on tRNA binding site. The rationale for using deacylated tRNA was that it is assumed to bind to the peptidyl donor site. We observed that tRNA has a strong protective effect on subunit modifications produced by LiCl: tRNA prevents subunit inactivation as measured by puromycin reaction and polyphenylalanine synthesis and it shifts the Li+/Mg2+ ratio value needed to reach 50% inactivation, from 60 to 250; it also prevents ribosomal protein and 5 S RNA release and large sedimentation changes of subunits, induced by LiCl. To explain the mechanism of 60 S subunit stabilization by tRNA, two hypotheses are considered: stabilization can be consequent on direct interaction of tRNA with specific proteins, or on maintenance on subunits of essential cations which are otherwise displaced by Li+, or both.

Animals↗
PubMed2005

The bipartite structure of the tRNA m1A58 methyltransferase from S. cerevisiae is conserved in humans.

Among all types of RNA, tRNA is unique given that it possesses the largest assortment and abundance of modified nucleosides. The methylation at N(1) of adenosine 58 is a conserved modification, occurring in bacterial, archaeal, and eukaryotic tRNAs. In the yeast Saccharomyces cerevisiae, the tRNA 1-methyladenosine 58 (m(1)A58) methyltransferase (Mtase) is a two-subunit enzyme encoded by the essential genes TRM6 (GCD10) and TRM61 (GCD14). While the significance of many tRNA modifications is poorly understood, methylation of A58 is known to be critical for maintaining the stability of initiator tRNA(Met) in yeast. Furthermore, all retroviruses utilize m(1)A58-containing tRNAs to prime reverse transcription, and it has been shown that the presence of m(1)A58 in human tRNA(3) (Lys) is needed for accurate termination of plus-strand strong-stop DNA synthesis during HIV-1 replication. In this study we have identified the human homologs of the yeast m(1)A Mtase through amino acid sequence identity and complementation of trm6 and trm61 mutant phenotypes. When coexpressed in yeast, human Trm6p and Trm61p restored the formation of m(1)A in tRNA, modifying both yeast initiator tRNA(Met) and human tRNA(3) (Lys). Stable hTrm6p/hTrm61p complexes purified from yeast maintained tRNA m(1)A Mtase activity in vitro. The human m(1)A Mtase complex also exhibited substrate specificity--modifying wild-type yeast tRNA(i) (Met) but not an A58U mutant. Therefore, the human tRNA m(1)A Mtase shares both functional and structural homology with the yeast tRNA m(1)A Mtase, possessing similar enzymatic activity as well as a conserved binary composition.

Amino Acid Sequence↗
PubMedDate not supplied

[Affinity modification of Escherichia coli ribosomes near the acceptor tRNA-binding site].

It was shown that Phe-tRNA Phe derivatives bearing arylazidogroups scattered statistically on N7 guanosine residues retain the ability to EF-Tu-dependent binding to E. coli ribosomes. UV-irradiation of the corresponding complex with the derivative of Phe-tRNA Phe located at A-site results in a specific modification of both ribosomal subunits to an approximately equal extent. It was found that proteins S9, S15, S16, S17, S18, S19 and L8/L9, L13, L15, L27 are labelled at A-site.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Purification and properties of a translation inhibitor from wheat germ.

A translation inhibitor from wheat germ has been purified more than 400-fold to apparent homogeneity. The inhibitor is a basic protein with a molecular weight of 30 000. This protein effectively blocks protein synthesis in animal cell-free extracts but does not affect protein synthesis in intact cells. Inhibition occurs at a ribosome to inhibitor molar ratio of 100:1, indicating an enzymic mechanism of action. The wheat germ protein inhibits the translation of endogenous mRNA, exogenous mRNA, and poly(uridylic acid) at a step in polypeptide chain elongation and without breakdown of the polysomes. Neither the aminoacylation reaction nor mRNA degradation is affected by the inhibitor. An interesting feature of the inhibition reaction is that it requires, in addition to the wheat germ inhibitor, both ATP and tRNA. The function of these two compounds in the inhibition is presently unknown since neither the hydrolysis of the beta,gamma-pyrophosphate bond of ATP nor a modification of the tRNA can be demonstrated during the reaction.

Kinetics↗
PubMed1993

A highly sensitive probe for guanine N7 in folded structures of RNA: application to tRNA(Phe) and Tetrahymena group I intron.

A nickel complex has been shown to promote conformation-specific oxidation of guanosine in polynucleotide RNA. In all cases, reaction was strictly dependent on the solvent exposure and surface properties of guanine N7. Modification of native tRNA(Phe) (yeast) was detected at G18, G19, G20, and Gm34 and concurred with predictions based on its crystal structure. Additional guanine derivatives became exposed to oxidation only after the tRNA unfolded in the absence of Mg2+. Reaction of the Tetrahymena group I intron RNA (L-21 ScaI) also compared favorably to its three-dimensional model by appropriately identifying guanosine residues in hairpin loops, duplex termini, and the essential cofactor binding site. These results complemented prior data generated by hydroxyl radical, and in combination they served to distinguish the solvent accessibility of sugar backbone and base positions in guanosine residues. Most importantly, this nickel complex exhibited greater selectivity than either dimethyl sulfate or RNase T1 for characterizing tRNA(Phe) and intron RNA.

Animals↗
PubMed1977

Alteration of the kinetic parameters for aminoacylation of Escherichia coli formylmethionine transfer RNA by modification of an anticodon base.

Treatment of Escherichia coli formylmethionine tRNA with 2 M sodium bisulfite, pH 7.0, in 10 mM MgCl2 at 25 degrees results in formation of uridine/bisulfite adducts at U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop. Two products, corresponding to the two diastereoisomers of 5,6-dihydrouridine-6-sulfonate, are formed at each reactive site in the tRNA. Although none of the modifications cause complete loss of methionine acceptor activity, the modified tRNA is amino-acylated at a reduced rate and has a decreased affinity for E. coli methionyl-tRNA synthetase. Aminoacylation of [35S]bisulfite-labeled tRNAfMet with a limiting amount of purified enzyme followed by separation of the acylated and unacylated molecules and structural analysis has shown that the presence of a specific diastereoisomer of the uridine/bisulfite adduct in the anticodon base U37 alters the kinetic parameters for aminoacylation of tRNAfMet.

Amino Acyl-tRNA Synthetases↗
PubMed1996

Mitochondrial import of a yeast cytoplasmic tRNA (Lys): possible roles of aminoacylation and modified nucleosides in subcellular partitioning.

The yeast tRNA(CUU)LYS is transcribed from a nuclear gene and then unequally redistributed between the cytosol (97-98%) and mitochondria (2-3%). We have optimized the conditions for its specific import into isolated mitochondria. However, only a minor fraction (about 0.5%) of the added tRNA was translocated into the organelles. An in vitro transcript, once aminoacylated, appeared to be a better import substrate than the natural tRNA which carries modified nucleosides. The tRNA is translocated across mitochondrial membranes in its aminoacylated form and remains relatively stable inside the organelle. Possible roles of aminoacylation, tRNA-protein interactions and nucleoside modification in subcellular partitioning of the tRNA are discussed.

Cell Fractionation↗
PubMed1996

Codon-reading specificity of an unmodified form of Escherichia coli tRNA1Ser in cell-free protein synthesis.

Unmodified tRNA molecules are useful for many purposes in cell-free protein biosynthesis, but there is little information about how the lack of tRNA post-transcriptional modifications affects the coding specificity for synonymous codons. In the present study, we prepared an unmodified form of Escherichia coli tRNA1Ser, which originally has the cmo5UGA anticodon (cmo5U = uridine 5-oxyacetic acid) and recognizes the UCU, UCA and UCG codons. The codon specificity of the unmodified tRNA was tested in a cell-free protein synthesis directed by designed mRNAs under competition conditions with the parent tRNA1Ser. It was found that the unmodified tRNA with the UGA anti-codon recognizes the UCA codon nearly as efficiently as the modified tRNA. The unmodified tRNA recognized the UCU codon with low, but detectable efficiency, whereas no recognition of the UCC and UCG codons was detected. Therefore, the absence of modifications makes this tRNA more specific to the UCA codon by remarkably reducing the efficiencies of wobble reading of other synonymous codons, without a significant decrease in the UCA reading efficiency.

Amino Acid Sequence↗
PubMed2026

Genes near tRNAs are enriched in translational machinery.

Transfer RNAs (tRNAs) are known for delivering amino acids to the growing polypeptide chain during translation. They can also influence gene expression, especially in times of nutrient starvation, through differential tRNA expression and modification. Transfer RNAs have a highly consistent cloverleaf structure, but relatively few known regulatory elements govern this conserved structure despite the 20 different standard isotypes. This study examines gene enrichment patterns near tRNA genes across 1149 fungal genomes. Genes enriched in proteasome regulation, ion transport, and rRNA were found to be significantly closer to tRNAs than other pathways. These results were consistent across KEGG overrepresentation analysis (ORA), KEGG gene set enrichment analysis (GSEA), and gene ontology (GO) analysis. Proteasome, ion transport, and RNA are all important aspects of protein production and regulation, suggesting that genes required for the synthesis and quality control of proteins, including tRNAs, are located near each other. Protein regulation is an energetically expensive process, and local co-regulation could increase efficiency and stress impacts on proteins.

RNA, Transfer↗
PubMed2000

Sequence, genomic organization and functional expression of the murine tRNA-specific adenosine deaminase ADAT1.

We have recently identified the first mammalian tRNA-specific adenosine deaminase human ADAT1, a member of the ADAR family of RNA editing enzymes. This protein is responsible for the first step of the unique A(37) to m(1)I(37) modification in eukaryotic tRNA(Ala). Here, we present the genomic structure of murine ADAT1 and the functional expression of mADAT1 cDNA. In mouse, as well as in human, ADAT1 is expressed from a single copy gene. The coding region of the mADAT1 gene is spread over nine exons, covering approximately 30kb of genomic DNA and encodes a protein of 499 amino acids. Overall, mADAT1 shares 81% nucleotide homology and 87.5% protein homology with the human ortholog. The recombinant mouse protein is active specifically and with a high efficiency on human tRNA(Ala) in vitro. Its genomic organization is compared to the structures of the sequence-related, pre-mRNA specific adenosine deaminases ADAR1 and ADAR2.

Adenosine Deaminase↗
PubMed1979

Studies on aminoacyl tRNA synthetases and transfer RNA in living Xenopus laevis oocytes.

The microinjection of transfer RNA into amphibian oocytes permits one to study under in vivo conditions the reactions that affect this important macromolecule. A comparative study has been carried out between the in vivo and in vitro specificity of the aminoacylation reacton. The results obtained show that modifications of the tRNA structure affect aminoacyl-tRNA synthetase recognition in the same fashion in both conditions. The in vivo aminoacylation was not affected by the presence of puromycin (0.5mM) or cycloheximide (0.1 mM) which completely inhibited oocyte protein synthesis. An interesting difference was obtained between the in vivo and in vitro aminoacylation of tRNA with regards to temperature requirements. While the in vivo reaction was optimal at 25 degrees and was totally inhibited at 37 degrees, the in vitro was optimal, at the latter temperature. The inhibition of the in vivo reaction at 37 degrees was not due to inactivation of the enzyme. The transfer of the amino acid moiety to nascent proteins was studied by measuring the transfer of radioactivity from injected (14C) phenylalanyl-tRNA into hot trichloroacetic acid precipitable material. It was found that 30% or more of the amino acid became incorporated into oocyte proteins and that this incorporation was due to direct transfer from the aminoacyl-tRNA and was inhibited by puromycin and cycloheximide.

Amino Acyl-tRNA Synthetases↗
PubMed1998

A novel wobble rule found in starfish mitochondria. Presence of 7-methylguanosine at the anticodon wobble position expands decoding capability of tRNA.

In the starfish mitochondrial (mt) genome, codons AGA and AGG (in addition to AGU and AGC) have been considered to be translated as serine. There is, however, only a single candidate mt tRNA gene responsible for translating these codons and it has a GCT anticodon sequence, but guanosine at the first position of the anticodon should base pair only with pyrimidines according to the conventional wobble rule. To solve this enigma, the mt tRNA GCUser was purified, and sequence determination in combination with electrospray liquid chromatography/mass spectrometry revealed that 7-methylguanosine is located at the first position of the anticodon. This is the first case in which a tRNA has been found to have 7-methylguanosine at the wobble position. It is suggested that methylation at N-7 of wobbling guanosine endows the tRNA with the capability of forming base pairs with all four nucleotides, A, U, G, and C, and expands the repertoire of codon-anticodon interaction. This finding indicates that a nonuniversal genetic code in starfish has been generated by base modification in the tRNA anticodon.

Animals↗