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At least 19 recordsLinked to original sources
PubMed1989

A human tRNA gene heterocluster encoding threonine, proline and valine tRNAs.

A cluster of three tRNA genes encoding a tRNA(UGUThr), a tRNA(UGGPro), and a tRNA(AACVal), and two Alu-elements occur in a 6.0-kb human DNA fragment. The tRNA(Thr) gene is 2.7-kb upstream from the tRNA(Pro) gene, which is separated by 367 bp from the tRNA(Val) gene. One Alu-element actually overlaps the tRNA(Val) gene and is of opposite polarity to all three tRNA genes. All three tRNA genes are accurately transcribed in a homologous HeLa cell extract, since the ribonuclease T1 fingerprints of the tRNA transcripts are consistent with the nucleotide sequences of the tRNAs. The upstream region flanking the tRNA(Thr) gene has two tracts of alternating purine/pyrimidine residues potentially capable of adopting the Z-DNA conformation, and presumptive binding sites for two RNA polymerase II transcription factors. The tRNA(Thr) gene apparently has a substantially higher in vitro transcriptional efficiency than the other two tRNA genes in this cluster, and a tRNA(GCCGly) gene from another human DNA segment. Deletion constructs of the tRNA(Thr) gene retaining 272, 168, and 33 bp of original 5'-flanking DNA had about the same in vitro transcriptional efficiency, whereas that of the construct with only 2 bp of 5'-flanking human DNA was drastically reduced. The tRNA(Thr) gene constructs with 272 and 168 bp of original 5'-flanking DNA apparently reduce the transcriptional efficiencies of the proline and glycine tRNA genes, implicating the upstream region from the tRNA(Thr) gene as being crucial for its high transcriptional efficiency.

Base Sequence↗
PubMed1995

The chloroplast trnP-trnW-petG gene cluster in the mitochondrial genomes of Beta vulgaris, B. trigyna and B. webbiana: evolutionary aspects.

The chloroplast trnP-trnW-petG gene cluster has been identified in the mitochondrial DNA (mtDNA) of sugar beet (Beta vulgaris). The chloroplast-derived trnW gene is transcribed in the mitochondria; the other two genes, however, do not seem to be transcribed. This gene cluster is also present in the mitochondrial genomes of two wild Beta species, B. trigyna and B. webbiana. Sugar beet and the two wild relatives share 100% sequence identity in the coding regions of both the mitochondrial trnP and trnW genes. On the other hand, the petG genes from the wild Beta mtDNAs were found to be disrupted either by a 5-bp duplication (B. trigyna) or by a deletion of the 5' region (B. webbiana). A data-base search revealed that a conserved sequence of 60 bp is present in the trnP-trnW intergenic region of the mitochondrial genomes of the three Beta species as well as in other higher plants, including wheat and maize, and that the conserved sequence is absent from the chloroplast counterpart. Our results thus favour the hypothesis of a monophyletic origin of the trnP-trnW-petG cluster found in the plant mitochondrial genomes examined.

Base Sequence↗
PubMed1989

Localization of three DNA segments encompassing tRNA genes to human chromosomes 1, 5, and 16: proposed mechanism and significance of tRNA gene dispersion.

The chromosomal locations of three cloned human DNA fragments encompassing tRNA genes have been determined by Southern analysis of human-rodent somatic cell hybrid DNAs with subfragments from these cloned genes and flanking sequences used as hybridization probes. These three DNA segments have been assigned to human chromosomes 1, 5, and 16, and homologous sequences are probably located on chromosome 14 and a separate locus on chromosome 1. These studies, combined with previous results, indicate that tRNA genes and pseudogenes are dispersed on at least seven different human chromosomes and suggest that these sequences will probably be found on most, if not all, human chromosomes. Short (8-12 nucleotide) direct terminal repeats flank many of the dispersed tRNA genes. The presence of these flanking repeats, combined with the dispersion of tRNA genes throughout the human genome, suggests that many of these genes may have arisen by an RNA-mediated retroposition mechanism. The possible functional significance of this gene dispersion is considered.

Animals↗
PubMed1991

Localization of a DNA segment encompassing four tRNA genes to human chromosome 14q11 and its use as an anchor locus for linkage analysis.

The chromosomal location of an 8.2-kb genomic fragment encompassing a cluster of four human tRNA genes has been determined by three complementary methods including Southern analysis of human/rodent somatic cell hybrids, in situ hybridization, and genetic linkage analysis. This tRNA cluster (TRP1, TRP2, and TRL1) is located near the T-cell receptor alpha (TCRA) locus at 14q11, and several RFLPs were detected at this site. These RFLPs and those at the TCRA and MYH7 (cardiac beta-MHC gene) loci have been used to type all informative members of the CEPH pedigrees. This has permitted ordering of these three gene loci and two anonymous probes (D14S26 and D14S25) in a 20-cM interval just below the centromere of chromosome 14. Based upon the chromosomal location and the polymorphisms at this site, one or more members of this gene cluster could serve as a useful anchor locus on chromosome 14.

Amino Acid Sequence↗
PubMed1995

Evidence for class-specific discrimination of a semiconserved base pair by tRNA synthetases.

Aminoacyl-tRNA synthetases have been divided into two classes based on the existence of two structurally distinct active sites. To date, few class-specific tRNA recognition features have been elucidated. High-resolution X-ray structures of representative class I and class II synthetases complexed to cognate tRNA substrates have been solved. In these structures, the class I enzyme approaches the end of the tRNA acceptor stem from the minor-groove side, while the class II synthetase approaches its cognate tRNA from the major-groove side. This distinction is reflected in the different initial sites (2'- or 3'-OH) of amino acid attachment. The role that the semiconserved G1.C72 terminal base pair plays in the aminoacylation of Escherichia coli tRNAs is probed in this in vitro study. We show here that class II alanyl-, prolyl-, and histidyl-tRNA synthetases are sensitive to changes at position 1 x 72. Previous work on class I synthetases and new data presented here with the valine-specific enzyme indicate that class I enzymes show little sensitivity to replacements of G1.C72. This work provides new evidence for class-specific differences in tRNA acceptor stem interactions that appear to be reflected not only in the initial site of aminoacylation but also in the mode of synthetase interaction with the semiconserved G1.C72 base pair proximal to the amino acid attachment site.

Alanine-tRNA Ligase↗
PubMed1993

A mitochondrial tRNA anticodon swap associated with a muscle disease.

We have identified an unusual mitochondrial (mt) tRNA mutation in a seven year-old girl with a pure myopathy. This G to A transition at mtDNA position 15990 changed the anticodon normally found in proline tRNAs (UGG) to the one found in serine tRNAs (UGA), and is the first pathogenic anticodon alteration described in a higher eukaryote. The mutant mtDNA was heteroplasmic (85% mutant) in muscle but was undetectable in white blood cells from the patient and her mother. Analysis of single muscle fibres indicated that mutant mtDNAs severely impaired mitochondrial protein synthesis and respiratory chain activity, but only when present at greater than 90%. The recessive behaviour of this mtDNA alteration may explain the patient's relatively mild clinical phenotype.

Anticodon↗
PubMed1989

Molecular analysis of the linear 2.3 kb plasmid of maize mitochondria: apparent capture of tRNA genes.

The nucleotide sequence and transcription pattern of the linear 2.3 kb plasmid of maize mitochondria was analyzed in order to elucidate its possible function in the organelle. The plasmid has 170 bp inverted repeats at its termini composed, in turn, of shorter repetitive sequences. An open reading frame within the plasmid is transcribed and can potentially specify a 33 kD product. In addition the plasmid contains two tRNA genes homologous to chloroplast sequences; the tRNApro(CAA) and the tRNAtrp(UGG). Both of the tRNA genes of the plasmid are transcribed, but apparently only the tRNAtrp is processed to the correct size. These tRNA sequences are found in the main mitochondrial genome of all higher plants tested, and in most maize relatives. An exception is the close maize relative Northern teosinte in which the tRNAtrp gene is also carried on a plasmid. These results suggest that the 2.3 kb plasmid has acquired the tRNA sequences from the main mitochondrial DNA. It is possible that the plasmid-encoded tRNAtrp gene is essential for organelle function thereby ensuring the maintenance of the plasmid in the mitochondrion.

Base Sequence↗
PubMed1990

In vitro processing of mitochondrial and plastid derived tRNA precursors in a plant mitochondrial extract.

A lysate of purified mitochondria of the higher plant Oenothera processes in vitro synthesized tRNA precursors to the mature tRNA size. In vitro synthesized transcripts containing genuine plant mitochondrial tRNAs and analogous RNAs from mitochondrial loci with plastid derived tRNA sequences are accurately processed by an RNAase P-like activity to yield the mature 5'-terminus. A four nucleotide deletion in the anticodon stem-loop structure, however, prevents processing. The results show that in vitro transcripts containing tRNAs from sequence fragments of plastid origin integrated in plant mitochondrial genomes can be processed correctly in plant mitochondria, if tRNA sequences and structures are intact.

Base Sequence↗
PubMed1995

Identity elements of tRNA(Thr) towards Saccharomyces cerevisiae threonyl-tRNA synthetase.

Identity elements of tRNA(Thr) towards Saccharomyces cerevisiae threonyl-tRNA synthetase were examined using in vitro transcripts. By mutation studies, a marked decrease in aminoacylation with threonine showed that the first base pair in the acceptor stem and the second and third positions of the anticodon are major identity elements of tRNA(Thr), which are essentially the same as those of Escherichia coli tRNA(Thr). Base substitution of the discriminator base, A73, by G73 or C73 impaired the threonine accepting activity, but not that by U73, suggesting that this position contributes to discrimination from other tRNAs possessing G73 or C73. No effects on aminoacylation were observed with substitutions at the second base pair in the acceptor stem. These are in contrast to E.coli tRNA(Thr) where the second base pair is required for the specific aminoacylation, with the discriminator base playing no roles. Of several mutations at the third base pair in the acceptor stem, only the G3-U70 mutation impaired the activity, suggesting that the G3-U70 wobble pair, the identity determinant of tRNAAla, acts as a negative element for threonyl-tRNA synthetase. These findings indicate that while the first base pair in the acceptor stem and the anticodon nucleotides have been retained as major recognition sites between S. cerevisiae and E.coli tRNA(Thr), the mechanism by which the synthetase recognizes the vicinity of the top of the acceptor stem seems to have diverged with the species.

Acylation↗
PubMed1992

A model for the aminoacyl-tRNA binding site of eukaryotic elongation factor 1 alpha.

Eukaryotic elongation factor 1 alpha (EF-1 alpha) binds all the aminoacyl-tRNAs except the initiator tRNA in a GTP-dependent manner. While the GTP binding site is delineated by the three GTP binding consensus elements, less is known about the aminoacyl-tRNA binding sites. In order to better understand this site, we have initiated cross-linking and protease mapping studies of the EF-1 alpha-GTP-aminoacyl-tRNA complex. Two different chemical cross-linking reagents, trans-diaminedichloroplatinum(II) and diepoxybutane, were used to cross-link four different aminoacyl-tRNA species to EF-1 alpha. A series of peptides were obtained, located predominantly in domains II and III. The ability of aminoacyl-tRNA to protect protease digestion sites was also monitored, and domain II was found to be protected from digestion by aminoacyl-tRNA. Last, an aminoacyl-tRNA analog with a reactive group on the aminoacyl side chain, N epsilon-bromoacetyl-Lys-tRNA, was cross-linked to EF-1 alpha. This reagent cross-liked to histidine 296 in a GTP-dependent manner and thus localizes the aminoacyl group adjacent to domain II. A model is developed for aminoacyl-tRNA binding to EF-1 alpha based on its similarity to the prokaryotic factor EF-Tu, for which an x-ray crystal structure is available.

Amino Acid Sequence↗
PubMed1991

Cloning and analysis of five mitochondrial tRNA-encoding genes from the fungus Beauveria bassiana.

Five mitochondrial (mt) tRNA genes from the filamentous fungus, Beauveria bassiana, were cloned and sequenced. The genes encoding the Val-, Ile-, Ser-, Trp- and Pro-accepting tRNAs were found clustered in the region 5' to the lrRNA-encoding gene. The genes were 64-77% homologous with the equivalent genes from other filamentous fungi, 49-58% to yeasts with the exception of the Val-accepting tRNA-encoding gene which was 76%, and only slightly homologous with Escherichia coli. The B. bassiana mt genetic code was found to be similar to that of other fungal mitochondria in that the UGA codon is used as a signal for Trp rather than as a stop codon. Transcript analysis has revealed that the genes present in tRNA cluster are transcribed and processed into tRNA-size products. Secondary structure models proposed for the gene products show that conservation of tRNA secondary structure also exists. The presence of a GGC sequence rather than a GGU sequence in the D-loop of the tRNA(Trp)-encoding gene is a feature unique to the B. bassiana mt tRNA. An unconventional G-A base pair present in the D-stem of the tRNA(Ser)-encoding gene is a feature conserved in the mt tRNA of other filamentous fungi. Comparison of the B. bassiana tRNA-encoding genes with those of two other filamentous fungi and two yeasts revealed that the differences between closely related species favoured transition-type mutations.

Base Sequence↗
PubMed1992

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein.

3T3 Cells↗