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At least 37 records · Page 2Linked to original sources
PubMed1992

Affinity electrophoretic detection of primary amino groups in nucleic acids: application to modified bases of tRNA and to aminoacylation.

Thiolation of primary amino groups in tRNA with the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate gives rise to species which are retarded during electrophoresis in organomercury-containing polyacrylamide gels. Since such amino groups occur, as far as is known, only as part of the modified bases 3-(3-amino-3-carboxypropyl)uridine and N-2-(5-amino-5-carboxypentyl)cytidine or as the alpha-amino group of aminoacylated tRNAs, this extension of the principle of affinity electrophoresis can be used for the detection and analysis of a specific functional group in both single tRNA species and in a mixed population. The strength of the interaction may be quantified and provides information on the chemical environment/conformation of the derivatized bases.

Amines↗
PubMed1996

Probing the higher order structure of RNA with peroxonitrous acid.

Potassium peroxonitrite (ONOOK) and [Fe(EDTA)]2- were used to analyze the influence of chemically entirely different hydroxyl radical sources on tRNA cleavage profiles. [Fe(EDTA)]2- gives rise to hydroxyl radicals via a Fenton-like reaction during the oxidation of chelated Fe2+, while ONOOK generates hydroxyl radicals via its conjugate acid (ONOOH) when adding a stable alkaline solution of ONOOK in samples buffered at neutral pH. [Fe(EDTA)]2- is known to induce oxidative strand scission at sugar moieties thought to be solvent accessible, while those residues located in the 'inside' of structured RNAs are protected. Although ONOOH is neutral and significantly smaller than the metal complex, both reagents generate the same protection pattern on tRNAs, suggesting that access of the commonly formed hydroxyl radical, rather than access of its source, is the determining factor when probing the higher order structure of RNA. Strong difference in reactivity is only seen at the modified 2-thiouridine S34 of tRNA(Lys3) which shows hyperreactivity towards ONOOK treatment. This particular reaction may require interaction between the peroxonitrite anion and the thiocarbonyl group of the base, since hyperreactivity is not observed when probing the dethiolated tRNA(Lys3).

Animals↗
PubMed1987

Analysis of a human gene cluster coding for tRNA(GAAPhe) and tRNA(UUULys).

A 13.8-kb fragment of human DNA isolated from a human lambda Charon-4A DNA library was found to contain four human tRNA genes. Nucleotide sequence analysis of approx. 3.7 kb of this segment of human DNA identified two lysine tRNA(UUU) genes identical in coding sequence to a previously reported human lysine tRNA gene [Roy et al., Nucl. Acids Res. 10 (1982) 7313-7322]. The other two tRNA genes were phenylalanine tRNA(GAA) genes, the first to be isolated from a mammalian source. These phenylalanine tRNA(GAA) genes were identical in sequence with the exception of a G/A polymorphism at coordinate 57. None of these tRNA genes contains introns. The tRNA(UUULys) and tRNA(GAAPhe) genes are organized in alternating order and are irregularly spaced, by intergenic regions of approx. 1.0, 2.6 and 5.0 kb, and randomly oriented. There was no evidence to indicate that any of these genes arose by gene duplication, since flanking sequence homology was limited to the putative RNA polymerase III termination signals in the 3'-flanking regions. A mature tRNA-sized product was identified following the transcription of each tRNA gene in a homologous in vitro transcription system. Interestingly, different levels of transcriptional activity of the three identical lysine tRNA genes were observed, suggesting modulation of tDNA expression by extragenic sequences. In addition, a minimum of eight regions of homology to Alu-type repetitive elements were detected in this human DNA fragment, one of which was located 53 bp upstream from a tRNA(GAAPhe) gene.

Base Sequence↗
PubMed1997

Both lumenal and cytosolic gating of the aqueous ER translocon pore are regulated from inside the ribosome during membrane protein integration.

Portions of the nascent chain are exposed to the lumen, the cytosol, or neither at different stages during the cotranslational integration of a protein into the ER membrane, as shown by compartment-specific collisional quenching of fluorophores incorporated into the polypeptide. The opening or closing of each end of the aqueous translocon pore is tightly controlled and occurs in a sequence that does not compromise the membrane's permeability barrier. Surprisingly, these structural changes at the membrane are effected by the transmembrane segment in the nascent protein from inside the ribosome. Thus, the ribosome, not the translocon, first recognizes the transmembrane segment and triggers long-range structural changes at the translocon that may be involved in shifting its function from translocation to integration.

Animals↗
PubMed2005

Mitochondrial myopathy and sideroblastic anemia (MLASA): missense mutation in the pseudouridine synthase 1 (PUS1) gene is associated with the loss of tRNA pseudouridylation.

A missense mutation in the PUS1 gene affecting a highly conserved amino acid has been associated with mitochondrial myopathy and sideroblastic anemia (MLASA), a rare autosomal recessive oxidative phosphorylation disorder. The PUS1 gene encodes the enzyme pseudouridine synthase 1 (Pus1p) that is known to pseudouridylate tRNAs in other species. Total RNA was isolated from lymphoblastoid cell lines established from patients, parents, unaffected siblings, and unrelated controls, and the tRNAs were assayed for the presence of pseudouridine (Psi) at the expected positions. Mitochondrial and cytoplasmic tRNAs from MLASA patients are lacking modification at sites normally modified by Pus1p, whereas tRNAs from controls, unaffected siblings, or parents all have Psi at these positions. In addition, there was no Pus1p activity in an extract made from a cell line derived from a patient with MLASA. Immunohistochemical staining of Pus1p in cell lines showed nuclear, cytoplasmic, and mitochondrial distribution of the protein, and there is no difference in staining between patients and unaffected family members. MLASA is thus associated with absent or greatly reduced tRNA pseudouridylation at specific sites, implicating this pathway in its molecular pathogenesis.

Anemia, Sideroblastic↗
PubMed1989

A human tRNA gene cluster encoding the major and minor valine tRNAs and a lysine tRNA.

A human genomic DNA clone hybridizing to mammalian valine tRNA(IAC) contained a cluster of three tRNA genes. Two valine tRNA genes with anticodons of AAC and CAC, encoding the major and minor cytoplasmic valine tRNA isoacceptors, respectively, and a lysine tRNA(CUU) gene were identified by Southern blot hybridization and DNA sequence analysis of a 7.1-kb region. At least nine Alu family members were interspersed throughout the 18.5-kb human DNA fragment, with three Alu elements in the intergenic region between the valine tRNA(AAC) gene and the lysine tRNA gene. Each of the five Alu family members in the sequenced region can be categorized into one of the four Alu subfamilies. The coding regions of all three tRNA genes contain characteristic internal split promoter sequences and typical RNA polymerase III termination signals in the 3'-flanking regions. The tRNA genes are accurately transcribed by RNA polymerase III in a HeLa cell extract, since the RNase T1 fingerprints of the mature-sized tRNA transcription products are consistent with the structural genes. The lysine tRNA(CUU) gene was transcribed only slightly more efficiently than the valine tRNA(CAC) gene in the homologous in vitro transcription system. Surprisingly, the valine tRNA(CAC) gene was transcribed about eightfold more efficiently than the valine tRNA(AAC) gene, implicating the presence of a modulatory element in the upstream region flanking the tRNA(CAC) gene.

Base Sequence↗
PubMed1990

Absence of methylation at HpaII sites in three human genomic tRNA sequences.

It has been known since the development of nearest neighbor analysis that the frequency of the dinucleotide CpG is markedly suppressed in vertebrate DNA (i.e. less than %C x %G). This suppression appears to be heterogeneous since it was shown some years ago that three vertebrate tRNA genes did not exhibit CpG suppression. We have analyzed 13 different human tRNA genes and found that they also do not exhibit CpG suppression. Because CpG suppression has been linked, to some extent at least, to the methylation-deamination process by which a methylated CpG is mutated to TpG, we investigated whether the lack of suppression of CpG in tRNAs could originate from an absence of methylation. Three human tRNA genes were selected from Genbank (lysine, Proline, and Phenylalanine) and examined for methylation at HpaII sites by polymerase chain reaction (PCR) and Southern blot analysis. The observed patterns were consistent with the absence of methylation at the seven HpaII sites analyzed in and around the tRNA genes, and we predict that the remaining CpGs in these genes will be unmethylated. Since GC-rich promoter regions also escape CpG suppression and since they are generally unmethylated, avoidance of methylation may be a general explanation for the absence of CpG suppression in selected regions of vertebrate genomes.

Base Sequence↗
PubMed1998

A liquid chromatography/electrospray mass spectrometric study on the post-transcriptional modification of tRNA.

Liquid chromatography/electrospray mass spectrometry is one of the rapidly developing techniques with which mass of large hydrophilic polymers such as proteins and nucleic acids can be determined precisely. The technique was applied to studies on the modifications of tRNAs. Various tRNA species purified from Escherichia coli were directly injected into a capillary reversed-phase column and the desalted and concentrated tRNAs were analyzed on-line with an electrospray mass spectrometer. In some cases, small but significant differences were noted between the theoretical and observed molecular masses, suggesting that there exist still unknown modifications. Under high resolution measurements, multiple peaks corresponding to species modified to a varying extent were resolved. To study the structures in detail, the isolated tRNA species were digested with ribonuclease T1, and the resulting mixture of fragments were analyzed by the same liquid chromatography/mass spectrometry. In this way, most of the fragments were easily identified solely from their masses, and the positions where the expected and real structures differ were revealed. The results obtained showed the presence of micro-heterogeneity among tRNAs and demonstrated at the same time the power of the hyphenated technique for the structural analysis on nucleic acids.

Anticodon↗
PubMed2000

Essential regions of the tRNA primer required for HIV-1 infectivity.

Human immunodeficiency virus (HIV), like all retroviruses, requires a cellular tRNA as a primer for initiation of reverse transcription. In a previous study, we demonstrated that an HIV-1 with a primer binding site complementary to yeast tRNA(Phe) (psHIV-Phe) was not infectious unless yeast tRNA(Phe) was supplied in trans. This unique in vivo complementation system has now been used to define the elements of the tRNA required for HIV-1 replication. Mutant tRNA(Phe) with deletions in TPsiC stem-loop, anticodon stem-loop or D stem-loop of the tRNA were generated and assessed for the capacity to rescue psHIV-Phe. Mutant tRNA(Phe) with disrupted TPsiC stem-loop did not rescue psHIV-Phe. In contrast, a mutant tRNA(Phe) without the D stem-loop was fully functional for the rescue. The tRNA anticodon stem-loop region was found to be important for efficient complementation. The results of our studies demonstrate for the first time the importance of specific structural and sequence elements of the tRNA primer for HIV-1 reverse transcription and define new targets for interruption of HIV-1 replication.

Base Sequence↗
PubMed1999

Screening by tRNA primer extension analysis of porcine kidney mRNA libraries defines a novel endogenous porcine retroviral long terminal repeat.

BACKGROUND: We have taken advantage of the common requirement of all eukaryotic retroelements for a specific tRNA primer to initiate DNA synthesis and applied a previously described in vitro screening methodology to the analysis of in vivo porcine tissues for transcriptionally active retroviral sequences. METHODS: A series of 18-base pair (bp) 3' tRNA oligomers complementary to established primer binding sites for a variety of vertebrate retroviruses, retrotransposons, and retroposons were applied to primer extension analysis of kidney poly(A) mRNA. Primer extension products are predicted to represent "strong stop" signals characteristic of the initial stages of retroviral transcription. RESULTS: Several extension products were cloned, sequenced, and analyzed as probes for screening the porcine genome for potentially active retroviral sequences. We used this strategy to identify and clone a 655-bp 5' long terminal repeat of a porcine retrovirus with significant homology to the simian sarcoma virus. This transcriptionally active virus has an 82-bp U5 region, a conserved AATAAA polyadenylation sequence, a 39-bp repeat reminiscent of other retroviral enhancers, and a unique glycine primer binding site. CONCLUSION: Our results suggest that tRNA primer cloning can effectively identify novel retroviral elements.

Animals↗
PubMed1988

Changing the identity of a tRNA by introducing a G-U wobble pair near the 3' acceptor end.

Although the genetic code for protein was established in the 1960's, the basis for amino acid identity of transfer RNA (tRNA) has remained unknown. To investigate the identity of a tRNA, the nucleotides at three computer-identified positions in tRNAPhe (phenylalanine tRNA) were replaced with the corresponding nucleotides from tRNAAla (alanine tRNA). The identity of the resulting tRNA, when examined as an amber suppressor in Escherichia coli, was that of tRNAAla.

Alanine↗
PubMed1989

Transfer RNA genes: landmarks for integration of mobile genetic elements in Dictyostelium discoideum.

In prokaryotes and eukaryotes mobile genetic elements frequently disrupt the highly conservative structures of chromosomes, which are responsible for storage of genetic information. The factors determining the site for integration of such elements are still unknown. Transfer RNA (tRNA) genes are associated in a highly significant manner with different putative mobile genetic elements in the cellular slime mold Dictyostelium discoideum. These results suggest that tRNA genes in D. discoideum, and probably tRNA genes generally in lower eukaryotes, may function as genomic landmarks for the integration of different transposable elements in a strictly position-specific manner.

Base Sequence↗
PubMed1994

Effects of alterations of primer-binding site sequences on human immunodeficiency virus type 1 replication.

The human immunodeficiency virus type 1 genomic RNA primer-binding site (PBS) sequence comprises 18 nucleotides which are complementary to those at the 3' end of the replication initiation primer tRNA(3Lys). To investigate the role of the PBS in viral replication, we either deleted the original wild-type PBS (complementary to tRNA(3Lys) or replaced it with DNA sequences complementary to either tRNA(1,2Lys) or tRNA(Phe). Transfection of COS cells with such molecular constructs yielded similar levels of viral progeny that were indistinguishable with regard to viral proteins and tRNA content. Virus particles derived from PBS-deleted molecular clones were noninfectious for MT-4, Jurkat, and CEM-T4 cells. However, infectious viruses were derived from constructs in which the PBS had been altered to sequences complementary to either tRNA(1,2Lys) or tRNA(Phe), although mutated forms showed significant lags in replication efficiency in comparison with wild types. Molecular analysis of reverse-transcribed DNA in cells infected by the mutated viruses indicated that both tRNA(1,2Lys) and tRNA(Phe) could function as primers for reverse transcription during the early stages of infection. Sequencing of full-length proviral DNA, obtained 6 days after infection, revealed the mutated PBS, indicating that a complete cycle of reverse transcription had occurred. During subsequent rounds of infection, reversion of the mutated PBS to wild-type sequences was observed, accompanied by increased production of viral gene products. Reversion to wild-type PBS sequences was confirmed both by specific PCR analysis, using distinct primer pairs, and by direct sequencing of amplified segments. We also performed endogenous in vitro reverse transcription experiments in which synthesis of minus-strand strong-stop viral DNA was primed from a synthetic RNA template containing a PBS complementary to various tRNA isoacceptors. These results showed that tRNA(3Lys) was a much more efficient primer of such reactions than either tRNA(1,2Lys) or tRNA(Phe).

Animals↗
PubMed2001

[Dementia/chorea].

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Adenosine Triphosphatases↗
PubMed1990

Reinvestigation of phosphorylation of tRNA in Escherichia coli.

This report shows the results of the reinvestigation of tRNA phosphorylation in E. coli. The phosphorylation did not occur on suppressor seryl-tRNA but occurred on other tRNA species. The activity of tRNA phosphorylation was found in E. coli extracts and partially purified. On DEAE-Sephadex A50 and PAGE gel, the phosphorylated-tRNA showed a pattern different from that the natural suppressor serine tRNA.

Chromatography, Ion Exchange↗