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At least 19 recordsLinked to original sources
PubMed2004

A cluster of metabolic defects caused by mutation in a mitochondrial tRNA.

Hypertension and dyslipidemia are risk factors for atherosclerosis and occur together more often than expected by chance. Although this clustering suggests shared causation, unifying factors remain unknown. We describe a large kindred with a syndrome including hypertension, hypercholesterolemia, and hypomagnesemia. Each phenotype is transmitted on the maternal lineage with a pattern indicating mitochondrial inheritance. Analysis of the mitochondrial genome of the maternal lineage identified a homoplasmic mutation substituting cytidine for uridine immediately 5' to the mitochondrial transfer RNA(Ile) anticodon. Uridine at this position is nearly invariate among transfer RNAs because of its role in stabilizing the anticodon loop. Given the known loss of mitochondrial function with aging, these findings may have implications for the common clustering of these metabolic disorders.

Adult↗
PubMed1992

Self-splicing introns in tRNA genes of widely divergent bacteria.

The organization of eukaryotic genes into exons separated by introns has been considered as a primordial arrangement but because it does not exist in eubacterial genomes it may be that introns are relatively recent acquisitions. A self-splicing group I intron has been found in cyanobacteria at the same position of the same gene (that encoding leucyl transfer RNA, UAA anticodon) as a similar group I intron of chloroplasts, which indicates that this intron predates the invasion of eukaryotic cells by cyanobacterial endosymbionts. But it is not clear from this isolated example whether introns are more generally present in different genes or in more diverse branches of the eubacteria. Many mitochondria have intron-rich genomes and were probably derived from the alpha subgroup of the purple bacteria (or Proteobacteria), so ancient introns might also have been retained in these bacteria. We describe here the discovery of two small (237 and 205 nucleotides) self-splicing group I introns in members of two proteobacterial subgroups, Agrobacterium tumefaciens (alpha) and Azoarcus sp. (beta). The introns are inserted in genes for tRNA(Arg) and tRNA(Ile), respectively, after the third anticodon nucleotide. Their occurrence in different genes of phylogenetically diverse bacteria indicates that group I introns have a widespread distribution among eubacteria.

Base Sequence↗
PubMed1974

Effect of L-methioninyl adenylate on the level of aminoacylation in vivo of tRNA(Met) from Escherichia coli K12.

In cells of E.coli K12 grown exponentially in minimal medium, tRNA(met), tRNA(leu) and tRNA(ile) are aminoacylated at 100%, 80% and 64%, respectively. On addition of L-methioninyl adenylate to the growth medium, one observes a specific deacylation of tRNA(met). When more than 35% of tRNA(met) is deacylated, growth rate is reduced and becomes proportional to the amount of methionyl-tRNA formed.

Adenosine Monophosphate↗
PubMed1989

Evolution of the mitochondrial genetic code. II. Reassignment of codon AUA from isoleucine to methionine.

The reassignment of codon AUA from isoleucine to methionine during mitochondrial evolution may be explained by the codon reassignment (capture) hypothesis without assuming direct replacement of isoleucine by methionine in mitochondrial proteins. According to this hypothesis, codon AUA would have disappeared from the reading frames of messenger RNA. AUA codons would have mutated mainly to AUU isoleucine codons because of constraints resulting from elimination of tRNA Ile with anticodon *CAU (in which *C is lysidine). Later, tRNA Met (CAU) would have undergone structural changes enabling it to pair with both AUG and AUA. AUA codons, formed by mutations of other codons, including AUG, would have reappeared and would have been translated as methionine.

Animals↗
PubMed2003

Allosteric regulation of tRNA import: interactions between tRNA domains at the inner membrane of Leishmania mitochondria.

Import of nucleus-encoded tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania involves recognition of specific import signals by the membrane-bound import machinery. Multiple signals on different tRNA domains may be present, and further, importable RNAs interact positively (Type I) or negatively (Type II) with one another at the inner membrane in vitro. By co-transfection assays, it is shown here that tRNA(Tyr) (Type I) transiently stimulates the rate of entry of tRNA(Ile) (Type II) into Leishmania mitochondria in transfected cells, and conversely, is inhibited by tRNA(Ile). Truncation and mutagenesis experiments led to the co-localization of the effector and import activities of tRNA(Tyr) to the D domain, and those of tRNA(Ile) to the variable region-T domain (V-T region), indicating that both activities originate from a single RNA-receptor interaction. A third tRNA, human tRNA(Lys), is imported into Leishmania mitochondria in vitro as well as in vivo. This tRNA has Type I and Type II motifs in the D domain and the V-T region, respectively, and shows both Type I and Type II effector activities. Such dual-type tRNAs may interact simultaneously with the Type I and Type II binding sites of the inner membrane import machinery.

Allosteric Regulation↗
PubMed1994

A tRNA gene mapping within the chloroplast rDNA cluster is differentially expressed during the development of Daucus carota.

In vivo analysis of expression of the chloroplast rDNA cluster during somatic embryogenesis of Daucus carota (D.carota) was performed by Northern-blot analysis with different DNA probes, spanning both the 16S rRNA gene, the 16S-23S rRNA spacer, which contains the two mosaic tRNA genes tRNA(Ile) and tRNA(Ala), and the region upstream of the 16S rRNA gene, where a tRNA(Val) maps. We show that expression both of the spacer tRNAs tRNA(Ile) and tRNA(Ala) is not significantly regulated during development whereas the amount of the transcript corresponding to tRNA(Val) is not detectable during early embryonic stages and progressively accumulates during late phases. Multiple transcription start sites have been identified upstream of the tRNA(Val) gene by S1 mapping analysis, which are activated late during the embryogenesis. These data indicate that developmental control mechanisms act on plastid gene expression during embryogenesis in carrot.

Base Sequence↗
PubMed2000

New chromatographic and biochemical strategies for quick preparative isolation of tRNA.

A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity. The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation. Optimal combinations are presented for three different cases. (i) tRNA(Phe) from Escherichia coli. This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC. (ii) tRNA(Ile) from E. coli: Aminoacylation increases the retention time for this tRNA in RP-HPLC. The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA(Ile) is recovered with high specific activity. (iii) tRNA(i)(Met) from Saccharomyces cerevisiae. The aminoacylated form of this tRNA is unstable. To increase stability, the aminoacylated form was formylated using E.coli: enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.COLI: The tRNA(i)(Met) recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation. These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.

Acylation↗
PubMed2001

Molecular organization of the ribosomal RNA transcription unit and the phylogenetic study of Zymomonas mobilis ZM4.

Previously we reported that Zymomonas mobilis ZM4 contains three ribosomal transcription units (rrnA to C operons) which are clustered around the 50 min region, PacI fragments 13 and 6, on the physical map of Z. mobilis ZM4 [Kang, H. L. and Kang, H. S. (1998) Gene 206, 223-228]. The physical map reveals that the rrnA gene set is located on the 76 kb PacI fragment 13. The complete nucleotide sequence of the rrnA gene set has been determined. The total number of nucleotides of the rrnA gene set is about 6,250 bp. The structural genes of the 16S, 23S and 5S rRNA code for the 1,478 nt, 2,786 nt and 121 nt RNA chains, respectively. The length of the spacer regions between the 16S and 23S rRNA genes, and between the 23S and 5S rRNA genes, are 606 bp and 101 bp, respectively. Two tRNA genes, Ile-tRNA and Ala-tRNA, are found between the 16S and 23S rRNA genes and a fMet-tRNA gene is identified downstream of the 5S rRNA gene. Thus, the molecular organization of this rrnA gene set is the order of 5'-16S rRNA-tRNAIle-tRNAAla-23S rRNA-5S rRNA-tRNAfMet-3'. The secondary structure models of 16S, 23S and 5S rRNA are proposed. The phylogenetic tree, based on the 16S rRNA sequence, was constructed by neighbor-joining and maximum-parsimony methods. Zymomonas belongs to a group in which Aqorbacterium, Rhodobacter and Sphingomonas are included.

Base Sequence↗
PubMed1990

A potato mitochondrial isoleucine tRNA is coded for by a mitochondrial gene possessing a methionine anticodon.

The nucleotide sequence of a potato mitochondrial tRNA(Ile) has been determined. This tRNA has a lysidine-like minor nucleotide in the first position of the anticodon (position 34). The corresponding mitochondrial gene has been cloned and sequenced. This gene has a CAT anticodon which corresponds to methionine. The C residue in the anticodon must therefore be post-transcriptionally modified. The mature tRNA has isoleucine-accepting activity but no methionine-accepting activity. This is the first report showing that in plant mitochondria a post-transcriptional modification could change the amino-acid specificity of a tRNA, as compared to that of the corresponding gene.

Anticodon↗
PubMed2000

Characterization of the three ribosomal RNA operons rrnA, rrnB, and rrnC, from Brucella melitensis.

The three Brucella melitensis ribosomal RNA operons rrnA, rrnB, and rrnC were characterized individually. Each locus consisted of the 16S rRNA gene (rrs), followed by an intergenic spacer containing the tRNA-Ile and tRNA-Ala genes, the 23S rRNA gene (rrl), an intergenic spacer devoid of tRNA genes, the 5S rRNA gene (rrf), and an f-Met tRNA gene. The DNA sequences were identical over a 6271bp region, diverging 594bp upstream of rrs and immediately downstream of the f-Met tRNA gene. The previously uncharacterized 23S rRNA genes each contained a 178bp insertion 130bp from the 5' end. The location of the insertion matched intervening sequences (IVSs) found in other Rhizobiaceae. However, the size and sequence of the Brucella IVS differed from all previously reported IVS sequences from bacteria. The IVS region was PCR-amplified from 20 Brucella isolates representing all known Brucella species and biovars. All isolates contained only the complete IVS fragment. We compared the IVS DNA sequences of rrlC from representative strains of each of the six known Brucella species. The data revealed that the sequences were identical and differed from the B. melitensis IVS sequences by a single base pair. In other bacterial species, the IVSs are associated with post-transcriptional processing of the 23S rRNA by RNase III. We found that the Brucella 23S rRNA was slightly smaller than the 23S rRNA of Escherichia coli, known to be devoid of IVS sequences.

Base Sequence↗
PubMed1994

Modulation of the suppression efficiency and amino acid identity of an artificial yeast amber isoleucine transfer RNA in Escherichia coli by a G-U pair in the anticodon stem.

The artificial amber suppressor corresponding to the major isoleucine tRNA from yeast (pVBt5), when expressed in E. coli, is a poor suppressor of the amber mutation lacIam181-Z. By analysing mutant forms, we could show that this was due to the presence of a U30-G40 wobble pair in the anticodon stem of the yeast tRNA and not to the level of the heterologously expressed tRNA. Efficient suppressors were obtained by restoring a normal U30-A40 or G30-C40 Watson-Crick pair. In vivo the mutant forms are exclusively charged by the bacterial lysyl-tRNA synthetase (LysRS), whereas the original yeast amber tRNA is charged at a low level by E. coli glutaminyl-tRNA synthetase (GlnRS) and LysRS. The inversion of the U30-G40 pair also induces a loss of the Gln identity. We conclude from these experiments that the U30-G40 base pair constitutes a negative determinant for LysRS interaction which operates either at the level of complex formation or at the catalytic step. As no direct contacts are seen between GlnRS and positions 30-40 of the complexed homologous tRNA, the U30-G40 pair of pVBt5 is believed to influence aminoacylation by GlnRS indirectly, probably at the level of the anticodon loop conformation by favouring an optimal apposition of the anticodon nucleotides with the protein.

Amino Acyl-tRNA Synthetases↗
PubMed1996

On the mechanism of leftward frameshifting at several hungry codons.

We have used lacZ reporter genes to assess leftward ribosome frameshifting on sequences containing the quadruplet U UUC followed by several different triplets coding for lysine, isoleucine, or leucine. Limitation for lysine-tRNA provokes leftward frameshifting when the slippery quadruplet is followed by either lysine codon aag or aaa, but not when followed by an isoleucine or leucine codon. Limitation for isoleucine provokes frameshifting when the quadruplet is followed by either isoleucine codon aua or auc, but not when it is followed by a lysine codon. We conclude that the quadruplet promotes shifting when the ribosome is stalled at any "hungry" codon immediately after it. Changing the quadruplet to U AGC, at which peptidyl-tRNA cognate to the AGC triplet will be mismatched at all three anticodon positions if it slips left, abolishes frameshifting when the ribosome is stalled at the next position. We conclude that the U UUC quadruplet promotes frameshifting by virtue of its ability to pair with a left-slipped peptidyl-tRNA. The frameshift promoted by isoleucine-tRNA limitation of the U UUC aua sequence was analyzed by amino acid sequencing of the protein product. It occurs through reading of the Cau histidine codon overlapping the hungry codon from the left. This result rules out a "simultaneous slippage" type of mechanism. It strongly suggests instead that starvation-promoted frameshifting occurs primarily by slippage of peptidyl-tRNA just upstream of the stall site, followed by decoding of the triplet overlapping the stall site from the left or 5' side. A secondary finding is that the last base of the "hungry" codon has a moderate effect on its shiftiness, aag being shiftier than aaa, and aua being shiftier than auc.

Amino Acid Sequence↗
PubMed1999

Pseudouridine synthetase Pus1 of Saccharomyces cerevisiae: kinetic characterisation, tRNA structural requirement and real-time analysis of its complex with tRNA.

Pseudouridine synthetase Pus1 from Saccharomyces cerevisiae is a multisite-specific enzyme that catalyses the formation of pseudouridine residues at different positions in several tRNA transcripts. Recombinant Pus1, tagged with six histidine residues at its N terminus was expressed in Escherichia coli and purified. Transcripts of yeast tRNAValand intronless yeast tRNAIlewere used as substrates to measure pseudouridine formation at position 27. The catalytic parameters Kmand kcatfor tRNAValand tRNAIlewere 420(+/-100) nM and 0.4(+/-0.1) min-1, 740(+/-100) nM and 0.5(+/-0.1) min-1, respectively. Pus1 possesses a general affinity for tRNA, irrespective of whether they are substrates. Its equilibrium dissociation constant ranges from 15 nM for the substrate yeast tRNAValand non-substrate yeast intronless tRNAPhe, to 150 nM for the substrate yeast intronless tRNAIle. The difference in the affinity for the different tRNA species is not reflected in the specific activity of the enzyme, indicating that the binding of Pus1 to tRNA is not the kinetically limiting step. The importance of tertiary base-pairs was investigated with several variants of yeast tRNAs. Although dispensable for activity, both the presence of a D-stem-loop and the presence of a G26.A44 base-pair, near the target uridine U27, are important elements for Pus1 tRNA high affinity recognition. The presence of a G26.A44 base-pair in tRNA increases its association constant rate with Pus1 (ka) by a factor of approximately 100, resulting in a decrease of the overall equilibrium dissociation constant (Kd). The dissociation rate (kd) is the same, independent of the presence of a G26.A44 base-pair in the tRNA. A model describing the interaction of Pus1 with tRNA is proposed.

Base Pairing↗
PubMed1999

Complementarity between 3' terminal nucleotides of tRNA and primer binding site is a major determinant for selection of the tRNA primer used for initiation of HIV-1 reverse transcription.

The initiation of reverse transcription of human immunodeficiency virus type 1 (HIV-1) exclusively utilizes tRNALys,3 as a primer. Previous studies have shown that HIV-1 could use alternative tRNAs, such as tRNAIle or tRNAHis, to initiate reverse transcription only if the primer binding site (PBS) was made complementary to the 3' terminal 18 nucleotides of the cognate tRNA. However, upon in vitro culture, the viruses with a PBS complementary to the alternative tRNAs rapidly reverted to generate a PBS complementary to tRNALys,3. To investigate the process of reversion, we have constructed defective proviral genomes that contain a PBS complementary to tRNAIle or tRNAHis. The genomes contain the gene for xanthine-guanosine phosphoribosyl transferase (gpt) in place of env. Cotransfection of these proviral genomes with a plasmid-encoding vesicular stomatitis virus G protein (VSV-G) results in viruses that undergo a single round of HIV-1 infection; successful infections are scored as cells resistant to the drug mycophenolic acid. Using this single-round infection system, we demonstrated that HIV-1 with a PBS complementary to tRNAIle or tRNAHis is three- to fivefold less efficient in replication as measured by production of drug-resistant cell colonies compared to the wild-type virus. These viruses predominantly used the cognate tRNA as primer in their initial round of replication, although we did obtain a single cell colony in which the PBS was complementary to tRNALys,3. Using an HIV-1 provirus with a PBS complementary to yeast tRNAPhe, we established a single-round infection system in which the infectivity of this mutant HIV-1 relies on transfected yeast tRNAPhe. The results of our studies suggest that the mechanism for selection of the tRNA primer for initiation of reverse transcription relies primarily on the complementarity between the tRNA primerthe PBS.

Binding Sites↗
PubMed1988

Characterization of phage-specific transfer RNA molecules coded by Vibrio eltor phage e4.

Transfer RNAs were isolated from phage e4-infected Vibrio eltor Mak 757 cells. These were aminoacylated with 14 individual 3H-labeled L-amino acids. Hybridization of these [3H]aminoacyl-tRNAs with phage e4 DNA revealed that the phage e4 encodes tRNAs for arginine, tryptophan, tyrosine, leucine, and isoleucine. Direct aminoacylation of phage-coded tRNA molecules isolated from phage DNA-RNA hybrids also confirmed this observation.

Bacteriophages↗
PubMed2001

Phylogenetic analysis of mitochondrial DNA in patients with an occipital stroke. Evaluation of mutations by using sequence data on the entire coding region.

Mitochondrial DNA (mtDNA) haplogroup U, defined by the polymorphism 12308A>G, may constitute a risk factor for an occipital stroke in migraine. We therefore identified 14 patients with an occipital stroke and with 12308A>G. We determined complete mtDNA coding region sequence for the patients and for population controls by conformation sensitive gel electrophoresis (CSGE) and direct sequencing. Sequence information was used to construct a phylogenetic network of mtDNA haplogroups U and K, which was found to be composed of subclusters U2, U4, U5 and a new subcluster U7, as well as cluster K. Five patients with a migrainous stroke belonged to subcluster U5 (P=0.006; Fisher's exact test). Many unique mutations were found among the patients with an occipital stroke including two tRNA mutations that have previously been suggested to be pathogenic. Analysis of mtDNA sequences by CSGE and comparison of the sequences through phylogenetic analysis greatly enhances the identification of mtDNA clusters in population and detection of mtDNA mutations in patients.

Adult↗
PubMed1999

Nucleotide determinants for tRNA-dependent amino acid discrimination by a class I tRNA synthetase.

The high accuracy of the genetic code relies on the ability of tRNA synthetases to discriminate rigorously between closely similar amino acids. While the enzymes can detect differences between closely similar amino acids at an accuracy of about 1 part in 100-200, a finer discrimination requires the presence of the cognate tRNA. The role of the tRNA is to direct the misactivated amino acid to a distinct catalytic site for editing where hydrolysis occurs. Previous work showed that three nucleotides at the corner of the L-shaped tRNA were collectively required. Here we show that each of these nucleotides individually contributes to the efficiency of editing. However, all are dispensable for the chemical step of hydrolysis. Instead, these nucleotides are required for translocation of a misactivated amino acid from the active site to the center for editing.

Acylation↗