RESUMO
Nuclear-encoded tRNAs are universally transcribed by RNA polymerase III (Pol-III) and contain intragenic promoters. Transcription of vertebrate tRNA(Sec) however requires extragenic promoters similar to Pol-III transcribed U6 snRNA. Here, we present a comparative analysis of tRNA(Sec) transcription in humans and the parasitic protozoa Trypanosoma brucei, two evolutionary highly diverged eukaryotes. RNAi-mediated ablation of Pol-II and Pol-III as well as oligo-dT induced transcription termination show that the human tRNA(Sec) is a Pol-III transcript. In T. brucei protein-coding genes are polycistronically transcribed by Pol-II and processed by trans-splicing and polyadenylation. tRNA genes are generally clustered in between polycistrons. However, the trypanosomal tRNA(Sec) genes are embedded within a polycistron. Their transcription is sensitive to α-amanitin and RNAi-mediated ablation of Pol-II, but not of Pol-III. Ectopic expression of the tRNA(Sec) outside but not inside a polycistron requires an added external promoter. These experiments demonstrate that trypanosomal tRNA(Sec), in contrast to its human counterpart, is transcribed by Pol-II. Synteny analysis shows that in trypanosomatids the tRNA(Sec) gene can be found in two different polycistrons, suggesting that it has evolved twice independently. Moreover, intron-encoded tRNAs are present in a number of eukaryotic genomes indicating that Pol-II transcription of tRNAs may not be restricted to trypanosomatids.
Assuntos
RNA Polimerase II/metabolismo , RNA de Transferência Aminoácido-Específico/genética , Trypanosoma brucei brucei/genética , Alfa-Amanitina/farmacologia , Sequência de Bases , Células HeLa , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Interferência de RNA , RNA Polimerase II/antagonistas & inibidores , RNA de Transferência Aminoácido-Específico/biossíntese , Transcrição Gênica/efeitos dos fármacosRESUMO
Objective: Graves' disease (GD) related hyperthyroidism (HT) has profound effects on metabolic activity and metabolism of macromolecules affecting energy homeostasis. In this study, we aimed to get a comprehensive understanding of the metabolic changes and their clinical relevance in GD children. Methods: We investigated serum substances from 30 newly diagnosed GD children and 30 age- and gender-matched healthy controls. We explored the metabolomics using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) analysis, and then analyzed the metabolomic data via multivariate statistical analysis. Results: By untargeted metabolomic analysis, a total of 730 metabolites were identified in all participants, among which 48 differential metabolites between GD and control groups were filtered out, including amino acids, dipeptides, lipids, purines, etc. Among these metabolites, 33 were detected with higher levels, while 15 with lower levels in GD group compared to controls. Pathway analysis showed that HT had a significant impact on aminoacyl-transfer ribonucleic acid (tRNA) biosynthesis, several amino acids metabolism, purine metabolism, and pyrimidine metabolism. Conclusion: In this study, via untargeted metabolomics analysis, significant variations of serum metabolomic patterns were detected in GD children.
Assuntos
Doença de Graves/metabolismo , Metabolômica , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Biomarcadores , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão , Feminino , Doença de Graves/genética , Humanos , Masculino , Redes e Vias Metabólicas , Purinas/metabolismo , Pirimidinas/metabolismo , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência Aminoácido-Específico/genética , Espectrometria de Massas em TandemRESUMO
The SGS1 gene of the yeast Saccharomyces cerevisiae encodes a DNA helicase with homology to the human Bloom's syndrome gene BLM and the Werner's syndrome gene WRN. The SRS2 gene of yeast also encodes a DNA helicase. Simultaneous deletion of SGS1 and SRS2 is lethal in yeast. Here, using a conditional mutation of SGS1, it is shown that DNA replication and RNA polymerase I transcription are drastically inhibited in the srs2Delta sgs1-ts strain at the restrictive temperature. Thus, SGS1 and SRS2 function in DNA replication and RNA polymerase I transcription. These functions may contribute to the various defects observed in Werner's and Bloom's syndromes.
Assuntos
DNA Helicases/fisiologia , Replicação do DNA , Proteínas Fúngicas/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transcrição Gênica , Síndrome de Bloom/genética , Códon , DNA Helicases/genética , DNA Fúngico/biossíntese , Proteínas Fúngicas/genética , Deleção de Genes , Genes Fúngicos , Humanos , Mutação , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Ribossômico/biossíntese , RNA de Transferência Aminoácido-Específico/biossíntese , RecQ Helicases , Saccharomyces cerevisiae/metabolismo , Síndrome de Werner/genéticaRESUMO
As originally postulated in Crick's Adaptor hypothesis, the faithful synthesis of proteins from messenger RNA is dependent on the presence of perfectly acylated tRNAs. The hypothesis also suggested that each aminoacyl-tRNA would be made by a unique enzyme. Recent data have now forced a revision of this latter point, with an increasingly diverse array of enzymes and pathways being implicated in aminoacyl-tRNA synthesis. These unexpected findings have far-reaching implications for our understanding of protein synthesis and its origins.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , RNA de Transferência Aminoácido-Específico/metabolismo , Aminoacil-tRNA Sintetases/classificação , Aminoacil-tRNA Sintetases/genética , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Evolução Molecular , Lisina-tRNA Ligase/classificação , Lisina-tRNA Ligase/metabolismo , Modelos Genéticos , Filogenia , Biossíntese de Proteínas , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência Aminoácido-Específico/genética , Especificidade por SubstratoRESUMO
RNA polymerase III catalyses the synthesis of tRNAs in eukaryotic organisms. Through combined biochemical and structural characterisation, multiple auxiliary factors have been identified alongside RNA Polymerase III as critical in both facilitating and regulating transcription. Together, this machinery forms dynamic multi-protein complexes at tRNA genes which are required for polymerase recruitment, DNA opening and initiation and elongation of the tRNA transcripts. Central to the function of these complexes is their ability to undergo multiple conformational changes and rearrangements that regulate each step. Here, we discuss the available biochemical and structural data on the structural plasticity of multi-protein complexes involved in RNA Polymerase III transcriptional initiation and facilitated re-initiation during tRNA synthesis. Increasingly, structural information is becoming available for RNA polymerase III and its functional complexes, allowing for a deeper understanding of tRNA transcriptional initiation. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.
Assuntos
RNA Polimerase III/metabolismo , RNA de Transferência/biossíntese , Iniciação da Transcrição Genética , Animais , Células Eucarióticas/metabolismo , Humanos , Modelos Genéticos , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas/genética , Subunidades Proteicas , RNA Polimerase III/química , RNA de Transferência/genética , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência Aminoácido-Específico/genética , Elongação da Transcrição Genética , Fatores de Transcrição/genéticaRESUMO
Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.
Assuntos
Biossíntese de Proteínas , Proteínas , RNA de Transferência Aminoácido-Específico/biossíntese , Animais , Sequência de Bases , Northern Blotting/métodos , Expressão Gênica , Isopenteniladenosina/genética , Isopenteniladenosina/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Selênio/metabolismo , SelenoproteínasRESUMO
We describe the genetically engineered overproduction of Escherichia coli tRNA(2Gln), its purification by high pressure liquid chromatography (HPLC), and its subsequent use in the growth of crystals of the E. coli glutaminyl-tRNA synthetase-tRNA(Gln) complex. The overproduced tRNA represents 60 to 70% of the total tRNA extracted from the engineered strain. A single anion exchange HPLC column is then sufficient to increase the purity of this isoacceptor to 90 to 95%. Crystals of this material complexed with the monomeric E. coli glutaminyl-tRNA synthetase enzyme were obtained by vapor diffusion from solutions containing sodium citrate as the precipitating agent. The crystals diffract to beyond 2.8 A resolution (1 A = 0.1 nm) and are of the orthorhombic space group C222(1) with unit cell parameters a = 240.5 A, b = 93.9 A, c = 115.7 A. Gel electrophoresis of dissolved crystals demonstrates the presence of both protein and tRNA.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Escherichia coli/metabolismo , RNA Bacteriano/biossíntese , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência de Glutamina/biossíntese , Cromatografia Líquida de Alta Pressão , Cristalização , Eletroforese em Gel de Poliacrilamida , RNA Bacteriano/isolamento & purificação , RNA de Transferência de Glutamina/isolamento & purificação , Difração de Raios XRESUMO
Sequencing of potato mitochondrial (mt) tRNA(Leu)(NAA) and of its cytosolic (cyt) counterpart revealed that these tRNAs are identical, except for a post-transcriptional modification: a Gm is present at position 18 in mt tRNA(Leu), instead of a G in cyt tRNA(Leu). Hybridization studies have shown that potato mt tRNA(Leu)(NAA) has a nuclear origin and must therefore be imported from the cytosol.
Assuntos
Processamento Pós-Transcricional do RNA , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência de Leucina/biossíntese , RNA/biossíntese , Solanum tuberosum/genética , Sequência de Bases , Citosol/análise , Fabaceae/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Plantas Medicinais , RNA Mitocondrial , Homologia de Sequência do Ácido NucleicoRESUMO
Dictyostelium tRNA genes can generally be expressed in vivo in yeast. Among tested Dictyostelium tRNA genes a tRNATrp gene containing a 13 bp intron is transcribed with particularly poor apparent efficiency and the intron is not removed. Elimination of the intron from the gene increases the amount of transcription products significantly. Splicing can only occur if minimal base-pairing of the anticodon with intron sequences is possible. Accumulation of tRNA gene transcripts decreases with the inability of intron splicing. Products of neither amber (UAG) nor opal (UGA) suppressor variants of the tRNATrp gene from Dictyostelium are able to suppress corresponding non-sense mutations in defined structural yeast genes. This also holds true for suppressor tRNA gene variants with precisely deleted intron regions.
Assuntos
Dictyostelium/genética , Genes Fúngicos , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência de Triptofano/biossíntese , Íntrons , Mutação , Splicing de RNA , RNA de Transferência de Triptofano/genética , Saccharomyces cerevisiae/genética , Transformação GenéticaRESUMO
Cell-free extracts of Escherichia coli and Bacillus subtilis catalyzed the tRNA-dependent, RNase A-sensitive formation of delta-aminolevulinic acid (ALA) from glutamate. Cell extracts prepared from cultures of E. coli grown under aerobic or anaerobic conditions had similar levels of ALA biosynthetic activity. Both the tRNA-stimulated conversion of glutamate to ALA and the conversion of glutamate-1-semialdehyde to ALA were inhibited by gabaculin. However, gabaculin had no effect on the growth of either E. coli or B. subtilis. The tRNA-dependent transformation of glutamate to ALA in E. coli and B. subtilis thus appears to be very similar to the pathway found in cyanobacteria, certain obligate anaerobic eubacteria, archaebacteria and in the chloroplasts of algae and higher plant species.
Assuntos
Ácido Aminolevulínico/metabolismo , Bacillus subtilis/metabolismo , Escherichia coli/metabolismo , Transferases Intramoleculares , Ácidos Levulínicos/metabolismo , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência de Ácido Glutâmico/biossíntese , Alanina/biossíntese , Alanina-tRNA Ligase/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Escherichia coli/genética , Via de Pentose Fosfato , Transaminases/metabolismoRESUMO
The effects of spermine have been studied on the aminoacylation reaction catalyzed by rat liver threonyl-tRNA synthetase. Spermine can not replace Mg2+ in this reaction. However, a stimulatory and synergistic effect was observed on the threonyl-tRNA formation, in the presence of spermine and suboptimal concentration of Mg2+. Other divalent cations like Ba2+, Ca2+, Mn2+ and Co2+ can substitute Mg2+ in the threonyl-tRNA formation, but in all these cases spermine had no significant effect. Spermine prevented the inhibitory effects caused by excess of ATP or tRNA on the aminoacylation reaction. Association constants were determined by equilibrium dialysis for the tRNA-spermine complex (Ka = 3.7 x 10(3) M-1) and by differential spectrophotometry for the ATP-spermine complex (Ka = 7.8 x 10(3) M-1). No enzyme-spermine complex could be detected by equilibrium dialysis. Some roles have been ascribed for the polyamine spermine in the stimulation of the threonyl-tRNA formation. ATP-spermine and tRNA-spermine can not function as substrates for the threonyl-tRNA synthetase, since Mg2+ is indispensable. The stimulatory effect by spermine is important considering the physiological concentration of Mg2+ in the tissues. Probably in vivo spermine would have a relevant role lowering the real Mg2+ concentration required in the aminoacylation reaction.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Fígado/enzimologia , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência de Treonina/biossíntese , Espermina/farmacologia , Treonina-tRNA Ligase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cátions Bivalentes , Cinética , Fígado/efeitos dos fármacos , Magnésio/farmacologia , Ratos , Espermina/metabolismo , Treonina/metabolismoRESUMO
Ochratoxin B (OTB), the dechloro-analogue of ochratoxin A (OTA), was studied separately and in combination with OTA on the aminoacylation of phenylalanine tRNA (tRNAPhe) catalysed by mice liver phenylalanyl-tRNA synthetase. OTB was neither a significant inhibitor of the reaction nor an antagonist of OTA. OTB was also assayed for its possible antagonistic effect on the in vivo protein synthesis inhibition caused by OTA in hepatoma tissue culture cells. No prevention of OTA inhibition could be found for OTB. It rather showed a slight additional inhibitory activity when mixed (100-180 microM) with low concentrations of OTA (40-60 microM). In conclusion, these results are not in favor of an antagonistic effect of OTB with respect to OTA action, at least on the level of cellular protein synthesis.
Assuntos
Neoplasias Hepáticas Experimentais/metabolismo , Ocratoxinas/farmacologia , Biossíntese de Proteínas , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência de Fenilalanina/biossíntese , Animais , Leucina/metabolismo , Camundongos , Ocratoxinas/antagonistas & inibidores , Ratos , Células Tumorais CultivadasRESUMO
In response to low (approximately 1 microM) levels of selenium, Escherichia coli synthesizes tRNA(Glu) and tRNA(Lys) species that contain 5-methylaminomethyl-2-selenouridine (mnm5Se2U) instead of 5-methylaminomethyl-2-thiouridine (mnm5S2U). Purified glutamate- and lysine-accepting tRNAs containing either mnm5Se2U (tRNA(SeGlu), tRNA(SeLys] or mnm5S2U (tRNA(SGlu), tRNA(SLys] were prepared by RPC-5 reversed-phase chromatography, affinity chromatography using anti-AMP antibodies and DEAE-5PW ion-exchange HPLC. Since mnm5Se2U, like mnm5S2U, appears to occupy the wobble position of the anticodon, the recognition of glutamate codons (GAA and GAG) and lysine codons (AAA and AAG) was studied. While tRNA(SGlu) greatly preferred GAA over GAG, tRNA(SeGlu) showed less preference. Similarly, tRNA(SGlu) preferred AAA over AAG, while tRNA(SeLys) did not. In a wheat germ extract--rabbit globin mRNA translation system, incorporation of lysine and glutamate into protein was generally greater when added as aminoacylated tRNA(Se) than as aminoacylated tRNA(S). In globin mRNA the glutamate and lysine codons GAG and AAG are more numerous than GAA and AAA, thus a more efficient translation of globin message with tRNA(Se) might be expected because of facilitated recognition of codons ending in G.
Assuntos
Escherichia coli/metabolismo , Compostos Organosselênicos , Biossíntese de Proteínas , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência de Ácido Glutâmico/biossíntese , RNA de Transferência de Lisina/biossíntese , Compostos de Selênio , Selênio/metabolismo , Sistema Livre de Células , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli/genética , RNA de Transferência/metabolismo , RNA de Transferência de Ácido Glutâmico/genética , RNA de Transferência de Ácido Glutâmico/isolamento & purificação , RNA de Transferência de Lisina/genética , RNA de Transferência de Lisina/isolamento & purificação , Ribonucleosídeos/análise , Selênio/análise , Óxidos de Selênio , Tiouridina/análise , Uridina/análiseRESUMO
Selenocysteinyl-tRNA(Sec), cysteinyl-tRNA(Cys), glutaminyl-tRNA(Gln), and asparaginyl-tRNA(Asn) in many organisms are formed in an indirect pathway in which a non-cognate amino acid is first attached to the tRNA. This non-cognate amino acid is then converted to the cognate amino acid by a tRNA-dependent modifying enzyme. The in vitro characterization of these modifying enzymes is challenging due to the fact the substrate, aminoacyl-tRNA, is labile and requires a prior enzymatic step to be synthesized. The need to separate product aa-tRNA from unreacted substrate is typically a labor- and time-intensive task; this adds another impediment in the investigation of these enzymes. Here, we review four different approaches for studying these tRNA-dependent amino acid modifications. In addition, we describe in detail a [32P]/nuclease P1 assay for glutaminyl-tRNA(Gln) and asparaginyl-tRNA(Asn) formation which is sensitive, enables monitoring of the aminoacyl state of the tRNA, and is less time consuming than some of the other techniques. This [32P]/nuclease P1 method should be adaptable to studying tRNA-dependent selenocysteine and cysteine synthesis.
Assuntos
Aminoácidos/biossíntese , RNA de Transferência/metabolismo , Radioisótopos de Carbono , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Redes e Vias Metabólicas , Radioisótopos de Fósforo , RNA de Transferência Aminoácido-Específico/biossíntese , Aminoacil-RNA de Transferência/biossíntese , RNA de Transferência de Asparagina/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismoRESUMO
The large ampullate glands of the orb-web spider, Nephila clavipes provide massive amounts of fibroin throughout the lifetime of the adult female. We have developed methods to culture the glands and manipulate their biosynthetic activity. This has allowed us to monitor a series of molecular events that precede silk production in glands excised from appropriately stimulated animals. In this paper, we demonstrate that prior to the transient dramatic production of fibroin, such glands accumulate large amounts of tRNAs cognate to the abundant amino acids in spider silk. One of these, alanine tRNA, appears to consist of two isoaccepting forms--one constitutive, and the other silkgland specific. Moreover, the silkgland-specific form appears to accumulate preferentially in response to stimulation. This phenomenon of tissue-specific tRNA production appears similar to that found in the silkglands of Bombyx mori, but the spider system has the unique property of permitting manipulation in vitro. Thus, it provides an unusual opportunity to study the mechanism of regulated tRNA synthesis.
Assuntos
RNA de Transferência de Alanina/biossíntese , RNA de Transferência Aminoácido-Específico/biossíntese , Aranhas/fisiologia , Animais , Northern Blotting , Fibroínas/biossíntese , Técnicas de Cultura de ÓrgãosRESUMO
Changes of tRNA species, as both relative percentage of total tRNA and absolute concentration, occur during liver cell proliferation induced by partial hepatectomy. Transfer RNAs which are abundant under quiescence are found to decrease during hepatocyte proliferation, and vice versa. One consequence of these changes is the differential expression of methionine-isoaccepting tRNA species. Initiator tRNA(Met) is present in scarce amounts under quiescent conditions and increases during cell-cycle progression. Elongator tRNA(Met) shows the opposite behavior. Both the quantitative and qualitative tRNA changes return to control levels as the liver returns to resting conditions. These changes might have mechanistic implications in modulating the protein synthesis required by cell proliferation. Moreover, the increase of initiator tRNA(Met) species might be necessary to translate protooncogene, growth factor, and receptor mRNAs, the translation of which is hindered by inhibitory AUG triplets upstream from the coding sequence. Thus the tRNA changes described herein could be involved in regulating translation of transcripts encoding cell-cycle associated proteins.
Assuntos
Divisão Celular/genética , Metionina/genética , RNA de Transferência Aminoácido-Específico/biossíntese , Animais , Cromatografia Líquida de Alta Pressão , Hepatectomia , Regeneração Hepática , Masculino , Ratos , Ratos Wistar , Fatores de TempoRESUMO
In the present study, a comprehensive, rapid and sensitive method for screening sequence variation of the human mitochondrial tRNA genes has been developed. For this purpose, the denaturing gradient gel electrophoresis (DGGE) technique has been appropriately modified for simultaneous mutation analysis of a large number of samples and adapted so as to circumvent the problems caused by the anomalous electrophoretic behavior of DNA fragments encoding tRNA genes. Eighteen segments of mitochondrial DNA (mtDNA), each containing a single uniform melting domain, were selected to cover all tRNA-encoding regions using the computer program MELT94. All 18 segments were simultaneously analyzed by electrophoresis through a single broad range denaturing gradient gel under rigorously defined conditions, which prevent band broadening and other migration abnormalities from interfering with detection of sequence variants. All base substitutions tested, which include six natural mutations and 14 artificially introduced ones, have been detected successfully in the present study. Several types of evidence strongly suggest that the anomalous behavior in DGGE of tRNA gene-containing mtDNA fragments reflects their tendency to form temporary or stable alternative secondary structures under semi-denaturing conditions. The high sensitivity of the method, which can detect as low as 10% of mutant mtDNA visually, makes it valuable for the analysis of heteroplasmic mutations.
Assuntos
DNA Mitocondrial/genética , Variação Genética , RNA de Transferência Aminoácido-Específico/genética , RNA/biossíntese , Linhagem Celular , Humanos , Ácidos Nucleicos Heteroduplexes/biossíntese , Mutação Puntual , RNA/genética , RNA Mitocondrial , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência de Leucina/biossíntese , RNA de Transferência de Leucina/genética , RNA de Transferência de Lisina/biossíntese , RNA de Transferência de Lisina/genéticaRESUMO
We used human tRNA(Tyr) precursor as a substrate to study self-excision of a pre-tRNA intron. This RNA was synthesized in vitro in a HeLa cell extract. It contains a 5' leader, an intron of 20 nucleotides and a 3' trailer. Self-cleavage of pre-tRNA(Tyr) occurs in 100 mM NH4OAc at a pH ranging from 6 to 8.5 in the presence of spermine, MgCl2 and Triton X-100 under conditions very similar to enzymatic intron excision. The reaction is temperature-dependent, relatively fast as compared to the enzyme-catalysed reaction and leads to fragments which resist further degradation. The detailed structure of all major and minor cleavage products was established by fingerprint analyses. Non-enzymatic cleavage occurs predominantly at the 3' splice site and to a minor extent at the 5' splice site. Other minor cleavage sites are located within the intron and in the 3' trailer. Putative 5' and 3' tRNA halves resulting from pre-tRNA(Tyr) self-cleavage are substrates for wheat germ RNA ligase, suggesting that the cleavage reaction yields 2',3'-cyclic phosphate and 5'-hydroxyl termini. Pre-tRNA splicing endonuclease is believed to cleave both the 5' and the 3' splice site. However, on the basis of our results we propose that this enzyme may support the formation of a pre-tRNA tertiary structure favourable for autocatalytic intron excision and impair unspecific self-cleavage.
Assuntos
Íntrons , Precursores de RNA/biossíntese , Splicing de RNA , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência de Tirosina/biossíntese , Sequência de Bases , Sistema Livre de Células , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas de Plantas/metabolismo , RNA Ligase (ATP)/metabolismo , Precursores de RNA/fisiologiaRESUMO
The 5'-terminal guanylate residue (G-1) of mature Escherichia coli tRNA(His) is generated as a result of an unusual cleavage by RNase P (Orellana, O., Cooley, L., and Söll, D. (1986) Mol. Cell. Biol. 6, 525-529). We have examined the importance of the unique acceptor stem structure of E. coli tRNA(His) in determining the specificity of RNase P cleavage. Mutant tRNA(His) precursors bearing substitutions of the normal base G-1 or the opposing, potentially paired base, C73, can be cleaved at the +1 position, in contrast to wild-type precursors which are cut exclusively at the -1 position. These data indicate that the nature of the base at position -1 is of greater importance in determining the site of RNase P cleavage than potential base pairing between nucleotides -1 and 73. In addition, processing of the mutant precursors by M1-RNA or P RNA under conditions of ribozyme catalysis yields a higher proportion of +1-cleaved products in comparison to the reaction catalyzed by the RNase P holoenzyme. This lower sensitivity of the holoenzyme to alterations in acceptor stem structure suggests that the protein moiety of RNase P may play a role in determining the specificity of the reaction and implies that recognition of the substrate involves additional regions of the tRNA. We have also shown that the RNase P holoenzyme and tRNA(His) precursor of Saccharomyces cerevisiae, unlike their prokaryotic counterparts, do not possess these abilities to carry out this unusual reaction.
Assuntos
Endorribonucleases/metabolismo , Proteínas de Escherichia coli , Precursores de RNA/metabolismo , RNA de Transferência Aminoácido-Específico/biossíntese , RNA de Transferência de Histidina/biossíntese , Escherichia coli/genética , Mutação , Conformação de Ácido Nucleico , RNA de Transferência de Serina/biossíntese , Ribonuclease P , Saccharomyces cerevisiae/genética , Relação Estrutura-AtividadeRESUMO
In this report, we have compared the changes in the production of tRNA(iMet) (initiator tRNA(Met] and tRNA(Asn), which occur during erythroid differentiation in the Friend erythroleukemia cell. The relative steady-state concentration of these two tRNAs (relative to the total tRNA population) was measured by aminoacylation. The results show that while the relative steady-state concentration of tRNA(iMet) changes very little in the cytoplasmic tRNA population, the relative concentration of tRNA(Asn) decreases during the first two days of differentiation and then undergoes an increase. This difference in the behavior of these two tRNAs is also seen when their relative concentrations in newly synthesized tRNA is examined. When tRNA is labeled with tritiated uridine for 24 h in vivo prior to isolation, the hybridization of this labeled tRNA to filter-bound tRNA genes shows that the relative concentration of tRNA(iMet) in newly synthesized tRNA changes very little, while the relative concentration of newly synthesized tRNA(Asn) again decreases through the first 2 days of differentiation, and then undergoes a smaller increase. Thus, the production of these two tRNAs appears to be independently regulated. Independent regulation of synthesis is also observed when examining the production of these two tRNAs in isolated nuclei. During erythroid differentiation, the relative synthesis of tRNA(iMet) (relative to total nuclear RNA synthesis) remains constant, while the relative synthesis of tRNA(Asn) undergoes periodic increases and decreases in value.