Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “tRNA”

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 721 records · Page 40Linked to original sources
PubMed1995

tRNA-guanine transglycosylase from Escherichia coli. Minimal tRNA structure and sequence requirements for recognition.

Previously, we have demonstrated that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is capable of utilizing an in vitro generated minihelix consisting of the anticodon stem and loop sequence of E. coli tRNA(Tyr) (Curnow, A. W., Kung, F. L., Koch, K. A., and Garcia, G. A. (1993) Biochemistry 32, 5239-5246). This suggests that the tRNA structural motifs necessary for recognition comprise a loop at the end of a short helix. To gain further insight into the structural requirements for TGT recognition, we have investigated the conformation of this minimal substrate. Thermal denaturation studies and kinetic analyses at 20 and 37 degrees C indicate that this minihelix is predominantly melted at 37 degrees C and that the melted conformation is not a substrate for TGT. This is confirmed by the determination that a non-helical analogue of the minihelix is not a substrate for TGT at either temperature. Two additional minihelices designed to be stable at 37 degrees C, ECYMH (a 4-base pair extension of the previous minihelix) and SCDMH (a yeast tRNA(Asp) analogue of ECYMH), were generated and characterized. Finally, several sequence mutants of SCDMH, focusing on the G30U40 base pair and U33G34U35 loop sequence, have been produced, and kinetic parameter determinations have been performed at 37 degrees C. Our results are consistent with a recent report (Nakanishi, S., Ueda, T., Hori, H., Yamazaki, N., Okada, N., and Watanabe, K. (1994) J. Biol. Chem. 269, 32221-32225) indicating that a UGU sequence in a 7-base loop is the minimal requirement for TGT recognition.

Anticodon↗
PubMed2003

Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria.

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.

Cloning, Molecular↗
PubMed1982

The tRNA-like structure at the 3' terminus of turnip yellow mosaic virus RNA. Differences and similarities with canonical tRNA.

The 3' terminus of TYMV RNA, which possesses tRNA-like properties, has been studied. A 3' terminal fragment of 112 nucleotides was obtained by cleavage with RNase H after hybridization of a synthetic oligodeoxynucleotide to the viral RNA. The accessibility of cytidine and adenosine residues was probed with chemical modification. Enzymatic digestion studies were performed with RNase T1, nuclease S1 and the double-strand specific RNase from the venom of the cobra Naja naja oxiana. A model is proposed for the secondary structure of the 3' terminal region of TYMV RNA comprising 86 nucleotides. The main feature of this secondary structure is the absence of a conventional acceptor stem as present in canonical tRNA. However, the terminal 42 nucleotides can be folded in a tertiary structure which bears strong resemblance with the acceptor arm of canonical tRNA. Comparison of this region of TYMV RNA with that of other RNAs from both the tymovirus group and the tobamovirus group gives support to our proposal for such a three-dimensional arrangement. The consequences for the recognition by TYMV RNA of tRNA-specific enzymes is discussed.

Base Sequence↗
PubMed1989

Aspartate and asparagine tRNA genes in wheat mitochondrial DNA: a cautionary note on the isolation of tRNA genes from plants.

We have identified genes encoding a "native" tRNA(Asp) (trnD-GTC) and a "chloroplast-like" tRNA(Asn) (trnN-GTT) on opposite strands and 633 bp apart within a sequenced 1640 bp RsaI restriction fragment of wheat mtDNA. The trnD gene has been found previously at a different location in wheat mtDNA (P.B.M. Joyce et al. (1988) Piant Mol. Biol. 11, 833-843); the duplicate copies of this gene are identical within the coding and immediate flanking regions (9 bp downstream and at least 68 bp upstream), after which obvious sequence similarity abruptly disappears. The trnN gene is identical to its homolog in maize ctDNA; continuation of sequence similarity beyond the coding region suggests that this gene originated as promiscuous ctDNA that is now part of the wheat mitochondrial genome. In the course of this work, we have encountered some unexpected similarities between tRNA gene regions from wheat mitochondria and other sources. Detailed analysis of these similarities leads us to suggest that trnN genes reportedly from petunia nuclear DNA (N. Bawnik et al. (1983) Nucleic Acids Res. 11, 1117-1122) and lupine mtDNA (B. Karpińska and H. Augustyniak (1988) Nucleic Acids Res. 16, 6239) are, in fact, from petunia mtDNA and lupine ctDNA, respectively, whereas a putative wheat nuclear tRNA(Ser) (trnS-TGA) gene (Z. Szwekowska-Kulińska et al. (1989) Gene 77, 163-167) is actually from wheat mtDNA. In these instances, it seems probable that the DNA samples used for cloning contained trace amounts of DNA from another sub-cellular compartment, leading to the inadvertent selection of spurious clones.

Base Sequence↗
PubMed1979

Cloning of chicken embryo tRNA genes using single stranded nucleosomal DNA highly enriched for tRNA complementary sequences.

DNA from chicken embryo nucleosome tetramers (about 760 base pairs in size) was enriched for tRNA genes by RPC-5 chromatography. The enriched DNA was hybridized with chicken embryo total tRNA and the hybridized DNA isolated utilizing a) avidinbiotin interaction, b) diazobenzyloxymethyl paper, and c) high temperature RPC-5 chromatography. The obtained single stranded DNA highly enriched for tRNA complementary sequences was hybridized with total DNA from nucleosome monomers (140--190 base pairs in size) and the excess of non hybridized monomer nucleosome DNA removed by Sepharose 4B chromatography. The hybrid molecules obtained were made fully double stranded by incubation with E. coli DNA polymerase I, DNA ligase, and exonuclease III. DNA was inserted into plasmid pBR322 by G-C joining procedure and the recombinant DNA used to transform the E. coli strain chi 1776. More than 70% of the transformants obtained hybridize to chicken embryo total tRNA.

Animals↗
PubMed1975

tRNA conformation and magnesium binding. A study of a yeast phenylalanine-specific tRNA by a fluorescent indicator and differential melting curves.

The binding of Mg2+ to tRNAPhe (yeast) in three conformational states was studied at 10, 30, 45, and 70 degrees C by the fluorescence indicator 8-hydroxyquinoline 5-sulphonic acid in the presence of 0.032 M monovalent cations (Na+). At temperatures below those characteristic for early melting (completely folded tRNA) the Scatchard plots are biphasic. They were well fitted by two classes of noninteracting binding sites with stability constants independent of temperature (KA = 9X10(4), KB = 6X10(3) M-1). In partially unfolded tRNA the strong binding process is co-operative. A single class of weak sites was found in the statistically coiled conformation at 70 degrees C (KB = 3.3X103 M-1). The total number of binding sites is 23 +/- 5; differences for the folded and unfolded conformations are smaller than 1. The influence of Mg2+ on the stability of the conformational elements of tRNAPhe (yeast) and its CCA-half (i.e. nucleotides 38--76) was determined by differential ultraviolet absorbance and depolarisation melting curves using the fluorescence of the Y base. Tertiary structure corresponding to early melting is stabilized by strongly bound Mg2+, whereas all other melting transitions are only influenced by Mg2+ bound at weak sites. The stability constants of tertiary structure obtained from the melting experiments can quantitatively be described by assuming that 5 +/- 1 non-interacting strong sites as characterized by the fluorescence titrations are converted to weak sites upon unfolding of the tertiary structure. Co-operative interaction of Mg2+ with the 5 strong sites in the folded conformation of tRNA can be ruled out. Strong binding of Mg2+ to completely folded tRNA does not produce a conformational transition changing ultraviolet absorbance, circular dichroism and sedimentation coefficient.

Binding Sites↗
PubMed1975

No correlation between native and denatured forms of tRNA(Trp) form Escherichia coli and the resistant and sensitive molecules characterised by phosphorolysis. Two classes of conformation characterised by phosphorolysis in both native and denatured tRNA(Trp).

Some tRNA molecules in solution are sensitive to attack by polynucleotide phosphorylase while others are resistant, even with pure species of tRNA. Further analysis of this behaviour has revealed an underlying microheterogeneity in tRNA structure. In order to clarify the relation between the sensitive and resistant classes of tRNA, and the native and denatured forms with respect to amino acid acceptance, the phosphorolysis of tRNATrp from Escherichia coli has been investigated. Native tRNATrp is similar to species examined previously: resistant and sensitive classes are observed and the sensitive proportion increases with temperature. At 20 degrees C both native and denatured tRNATrp are stable under phosphorolysis conditions, and denaturated tRNATrp is found also to possess resistant and sensitive classes. About 10% of both native and denatured tRNATrp is rapidly phosphorolysed at 20 degrees C, but the rate of conversion of resistant denatured tRNATrp to the sensitive class is about twice as fact as for the native form. Thus it can be concluded that the sensitive molecules of tRNATrp attacked by polynucleotide phosphorylase are not due to denaturation.

Drug Stability↗
PubMed1988

Transformation of Drosophila melanogaster with a suppressor tRNA gene (Sup3e tRNA(SerUGA)) from Schizosaccharomyces pombe.

P-element mediated transformation was utilized to introduce a suppressor tRNA gene (Sup3e tRNA(UGASER)) from Schizosaccharomyces pombe into Drosophila melanogaster. Thirteen independently transformed lines were characterized as to the number of cytological locations of the transposons. It was ascertained that the suppressor tRNA gene of interest was introduced into each transformed strain. The helper P element used (p pi 25.1) allows further transposition to occur, and it was determined that from one to seven copies of the heterologous tRNA(UGASER) gene per strain were present among the respective transformed strains. The number of transposons per transformed line was established by in situ hybridization to salivary gland chromosomes as well as by Southern hybridization analyses and there was good agreement in the totals determined by these two techniques.

Animals↗
PubMed2001

tRNA recognition by tRNA-guanine transglycosylase from Escherichia coli: the role of U33 in U-G-U sequence recognition.

In eubacteria, the biosynthesis of queuine, a modified base found in the wobble position (#34) of tRNAs coding for Tyr, His, Asp, and Asn, occurs via a multistep pathway. One of the key enzymes in this pathway, tRNA-guanine transglycosylase (TGT), exchanges the genetically encoded guanine at position 34 with a queuine precursor, preQ1. Previous studies have identified a minimal positive RNA recognition motif for Escherichia coli TGT consisting of a stable minihelix that contains a U-G-U sequence starting at the second position of its seven base anticodon loop. Recently, we reported that TGT was capable of recognizing the U-G-U sequence outside of this limited structural context. To further characterize the ability of TGT to recognize the U-G-U sequence in alternate contexts, we constructed mutants of the previously characterized E. coli tRNA(Tyr) minihelix. The U-G-U sequence was shifted to various positions within the anticodon loop of these mutants. Characterization of these analogs demonstrates that in addition to the normal U33G34U35 position, TGT can also recognize the U34G35U36 analog (UGU(+1)). The other analogs were not active. This indicates that the recognition of the U-G-U sequence is not strictly dependent upon its position relative to the stem. In E. coli, the full-length tRNA with a U34G35U36 anticodon sequence is one of the isoacceptors that codes for threonine. We found that TGT is able to recognize tRNA(Thr(UGU)) but only in the absence of a uridine at position 33. U33, an invariant base present in all tRNAs, has been shown to strongly influence the conformation of the anticodon loop of certain tRNAs. We find that mutation of this base confers on TGT the ability to recognize U34G35U36, and suggests that loop conformation affects recognition. The fact that the other analogs were not active indicates that although TGT is capable of recognizing the U-G-U sequence in additional contexts, this recognition is not indiscriminate.

Anticodon↗
PubMed1991

Structural and sequence elements important for recognition of Escherichia coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem.

We show that the structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. This conclusion is based on mutagenesis of the E. coli initiator tRNA gene followed by measurement of kinetic parameters for formylation of the mutant tRNAs in vitro and function in protein synthesis in vivo. The first base pair found at the end of the amino acid acceptor stem in all other tRNAs is replaced by a C.A. "mismatch" in E. coli initiator tRNA. Mutation of this C.A. to U:A, a weak base pair, or U.G., a mismatch, has little effect on formylation, whereas mutation to C:G, a strong base pair, has a dramatic effect lowering Vmax/Kappm by 495-fold. Mutation of the second basepair G2:C71 to U2:A71 lowers Vmax/Kappm by 236-fold. Replacement of the third base-pair C3:G70 by U3:A70, A3:U70, or G3:C70 lowers Vmax/Kappm by about 67-, 27-, and 30-fold, respectively. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and T psi C stem have little or no effect on formylation.

Acyltransferases↗
PubMed1990

Assignments of the iminoproton resonances of Bombyx mori tRNA(UCCGly) and the comparison of its structure and stability with those of tRNA(GCCGly).

Most of the iminoproton resonances in the 1H-NMR spectrum of Bombyx mori tRNA(UCCGly) have been assigned by the sequential NOEs. Any peak which indicates the presence of the tertiary GC base pair between the D and T loops could not be detected. The effects of temperature and the addition of magnesium ions and spermine on the 1H-NMR spectrum of this tRNA were examined. From the temperature change, it was found that the acceptor stem and the D stem in Bombyx mori tRNA(UCCGly) are equally stable even in the absence of magnesium, which is different from tRNA(GCCGly) where the D stem is not so stable.

Animals↗
PubMed1989

Recognition of tRNA by the enzyme ATP/CTP:tRNA nucleotidyltransferase. Interference by nucleotides modified with diethyl pyrocarbonate or hydrazine.

Treatment of tRNA with diethyl pyrocarbonate or hydrazine prior to incubation with the enzyme ATP/CTP:tRNA nucleotidyltransferase and [alpha-32P]ATP results in exclusion of modified bases from labeled molecules. Purines modified with diethyl pyrocarbonate, which interfere with enzyme recognition, cluster at the corner of the tRNA molecule, where the D- and psi-loops are juxtaposed in all 15 tRNAs used in this study. When the enzyme is isolated from Escherichia coli, few other sites of interference are evident near the 3'-end; when the homologous enzyme from yeast is used, more exclusions are apparent near the 3'-end. Modification of uridines with hydrazine has no effect on interaction with the enzyme, except for one uridine near the 3'-end of tRNA(Gly). Interference of enzyme activity by modified bases can be overcome by longer incubation times or increased concentrations of enzyme.

Diethyl Pyrocarbonate↗
PubMedDate not supplied

A mouse tRNA pseudogene derived from a tRNA(Trp) coding sequence.

The identification and characterization of a mouse tRNA(Trp) pseudogene is reported. A synthetic oligonucleotide (31 mer), identical with the 3' half of a tRNA(Trp), was used to examine three mouse lambda clones that are known to contain clusters of tRNA genes. A fragment of one of these lambda clones strongly hybridizes to the oligonucleotide; the sequence of this region shows the presence of a gene significantly homologous to the chick tRNA(Trp). However two point mutations and a three base deletion prevent the folding of a possible transcript of this gene. The presence of a conserved promoter sequence for RNA polymerase III, that should allow the transcription of this gene, does not ensure the transcription of the gene, at least in our in vitro system.

Animals↗
PubMed1988

The inhibitory effect of oligodeoxynucleotides complementary to different fragments of tRNA structure on the enzymatic binding of Phe-tRNA to poly-U-programmed eukaryotic ribosomes.

The effect of several oligodeoxynucleotides complementary to the fragments of yellow lupin tRNA(Phe) was tested in the aminoacylation of tRNA(Phe) and in the binding of Phe-tRNA(Phe) to poly-U-programmed eukaryotic ribosomes. Oligonucleotides tested in the aminoacylation test did not give any inhibition. Monomers and dimers did not have any significant influence on the binding assay, either. A different percentage of inhibition of the binding of Phe-tRNA to ribosomes has been observed for oligonucleotides. Heptamer complementary to the anticodon loop gave 100% inhibition of the binding reaction. However, the oligonucleotides complementary to both the anticodon loop and stem and longer than the heptamer were much less effective inhibitors. A high inhibitory effect was also observed for trimers and for the decamer complementary to the D-loop and CCA-end.

Base Sequence↗
PubMed1978

tRNA isopentenyltransferase from Zea mays L. Characterization of the isopentenylation reaction of tRNA, oligo (A) and other nucleic acids.

The extraction and purification of the tRNA isopentenyltransferase from maize root tips and kernels are reported. The relative amounts of this enzyme in different organs of maize have been determined. Root tips have the highest enzyme activities, followed by kernels and young leaves. Old leaves exhibit very low activity. The molecular mass of the monomeric enzyme was determined to be 57000 - 63000 dalton. pH and Mg2 optima are in full agreement with the data reported for the enzymes from yeast and Escherichia coli. Spermine enhances the isopentenylation of tRNA from kernels. The "Km" values of KMnO4-treated or untreated bulk tRNA from yeast and maize root tips and kernels were in the range of 10 to 34 micrometer while the Km values of of single species like tRNASer4 from rat liver and tRNATyrKMnO4 from E. coli were 1.1 and 6.6 micrometer, respectively. The tRNA isopentenyltransferase from maize root tips and kernels catalyzes the incorporation of 2-isopentenyl groups into (Ap)3-7A, endogenous bulk oligonucleotos from maize root tips and kernels, and into poly(A), RNA from MS-2 phages and, to a very low extent, into adenosine. The Km values of (Ap)3-7 A varied in the range of 250 to 750micrometer. Although oligonucleotides have less affinity to the enzyme, the formation of i6A within oligonucleotides may occur in vivo, due to their higher concentrations in cells.

Adenosine↗
PubMed1995

The ribosomal environment of tRNA: crosslinks to rRNA from positions 8 and 20:1 in the central fold of tRNA located at the A, P, or E site.

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine ("acp3U") at position 20:1 of lupin tRNAMet was coupled to a photoreactive diazirine derivative. Similarly, the 4-thiouridine at position 8 of Escherichia coli tRNAPhe was modified with an aromatic azide. Each of the derivatized tRNAs was bound to E. coli ribosomes in the presence of suitable mRNA analogues, under conditions specific for the A, P, or E sites. After photoactivation of the diazirine or azide groups, the sites of crosslinking from the tRNAs to 16S or 23S rRNA were analyzed by our standard procedures, involving a combination of ribonuclease H digestion and primer extension analysis. The crosslinked ribosomal proteins were also identified. The results for the rRNA showed a well-defined series of crosslinks to both the 16S and 23S molecules, the most pronounced being (1) an entirely A-site-specific crosslink from tRNA position 20:1 to the loop-end region (nt 877-913) of helix 38 of the 23S RNA (a region that has not so far been associated at all with tRNA binding), and (2) a largely P-site-specific crosslink from tRNA position 8 to nt 2111-2112 of the 23S RNA (nt 2112 being a position that has previously been identified in footprinting studies as belonging to the ribosomal E site). The data are compared with results from a parallel study of crosslinks from position 47 (also in the central fold of the tRNA), as well as with previously published crosslinks from the anticodon loop (positions 32, 34, and 37) and the CCA-end region (position 76, and the aminoacyl residue).

Base Sequence↗
PubMed1997

Mitochondrial tRNA(Gln) and tRNA(Thr) gene variants in Parkinson's disease.

A number of neurodegenerative diseases including Parkinson's disease (PD) have been shown to be associated with polymorphic variants of mitochondrial DNA. The A4336G mutation in the tRNA(Gln) gene was reported to occur at a higher frequency in individuals with Parkinson's disease and Alzheimer's disease (AD) than in age-matched controls. Similarly, we recently noted an elevated frequency of alterations at positions 15927 and 15928 in the tRNA(Thr) gene, resulting in the loss of a HpaII site, in patients suffering from multiple sclerosis (MS) with severe optic involvement. Here we report on the result of screening 100 PD patients and 100 age- and sex-matched controls for the presence of the A4336G mutation and the loss of the HpaII site in the tRNA(Thr) gene. Our result shows that loss of the HpaII site is significantly more frequent in patients than in controls. In contrast, we were not able to detect a difference in the frequency of the A4336G mutation in the tRNA(Gln) gene between patients and controls.

Aged↗
PubMed1985

Isolation of gram quantities of isoleucyl-tRNA synthetase from an overproducing strain of Escherichia coli and its use for purification of cognate tRNA.

The ileS gene coding for isoleucyl-tRNA synthetase was cloned on a runaway-replication plasmid. From the cells harboring the plasmid, gram quantities of the synthetase were isolated using two column procedures. The synthetase was used for the purification of cognate tRNA. Isoleucine tRNAGAU of greater than 90% purity was easily isolated by taking advantage of a specific complex formation of the synthetase with cognate tRNA.

Amino Acyl-tRNA Synthetases↗