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At least 19 recordsLinked to original sources
PubMed1996

Arginine aminoacylation identity is context-dependent and ensured by alternate recognition sets in the anticodon loop of accepting tRNA transcripts.

Yeast arginyl-tRNA synthetase recognizes the non-modified wild-type transcripts derived from both yeast tRNA(Arg) and tRNA(Asp) with equal efficiency. It discriminates its cognate natural substrate, tRNA(Arg), from non-cognate tRNA(Asp) by a negative discrimination mechanism whereby a single methyl group acts as an anti-determinant. Considering these facts, recognition elements responsible for specific arginylation in yeast have been searched by studying the in vitro arginylation properties of a series of transcripts derived from yeast tRNA(Asp), considered as an arginine isoacceptor tRNA. In parallel, experiments on similar tRNA(Arg) transcripts were performed. Unexpectedly, in the tRNA(Arg) context, arginylation is basically linked to the presence of residue C35, whereas in the tRNA(Asp) context, it is deeply related to that of C36 and G37 but is insensitive to the nucleotide at position 35. Each of these nucleotides present in one host, is absent in the other host tRNA. Thus, arginine identity is dependent on two different specific recognition sets according to the tRNA framework investigated.

Anticodon↗
PubMed2002

A one-step method for in vitro production of tRNA transcripts.

Sequencing of a large number of microbial genomes has led to the discovery of new enzymes involved in tRNA biosynthesis and tRNA function. Preparation of a great variety of RNA molecules is, therefore, of major interest for biochemical characterization of these proteins. We describe a fast, cost-effective and efficient method for in vitro production of tRNA transcripts. T7 RNA polymerase requires a double-stranded DNA promoter in order to initiate transcription; however, elongation does not require a double-stranded DNA template. A partially double-stranded transcription template formed by annealing of a short oligonucleotide, complementary to the T7 promoter, to a larger oligonucleotide is shown to be a good substrate for in vitro transcription. This method allows rapid production of a variety of tRNA transcripts which can be aminoacylated well. This eliminates the need for cloning of tRNA genes, large-scale plasmid preparation and enzymatic digestion.

DNA↗
PubMed1998

Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity.

A limitation for a universal use of T7 RNA polymerase for in vitro tRNA transcription lies in the nature of the often unfavorable 5'-terminal sequence of the gene to be transcribed. To overcome this drawback, a hammerhead ribozyme sequence was introduced between a strong T7 RNA polymerase promoter and the tDNA sequence. Transcription of this construct gives rise to a 'transzyme' molecule, the autocatalytic activity of which liberates a 5'-OH tRNA transcript starting with the proper nucleotide. The method was optimized for transcription of yeast tRNA(Tgammar), starting with 5'-C1, and operates as well for yeast tRNA(Asp) with 5'-U1. Although the tRNAs produced by the transzyme method are not phosphorylated, they are fully active in aminoacylation with k(cat) and Km parameters quasi identical to those of their phosphorylated counterparts.

Biochemistry↗
PubMed1993

Random-splitting of tRNA transcripts as an approach for studying tRNA-protein interactions.

Location of phosphodiester bonds essential for aminoacylation of bovine tRNA(Trp) was identified using a randomly cleaved transcript synthesized in vitro. It was found that cleavage of phosphodiester bonds after nucleotides in positions 21, 22, 36-38, 57-59, 62 and 64 were critical for aminoacylation capacity of tRNA(Trp)-transcript. These cleavage sites were located in the regions of tRNA molecule protected by the cognate synthetase against chemical modification and in the regions presumably outside the contact area as well. These results indicate that for maintenance of aminoacylation ability the intactness of the certain regions of the tRNA backbone structure is necessary. Random splitting of non-modified RNA with alkali followed by separation of active and inactive molecules and identification of cleavage sites developed in this work may become a general approach for studying the role of RNA covalent structure in its interaction with proteins.

Acylation↗
PubMed1988

Interaction of tRNA transcription factors with satellite I DNA from Xenopus laevis.

A cloned repeat of Xenopus laevis satellite I DNA was tested for the ability to form stable complexes with tRNA transcription factors in vitro. In template exclusion studies, the satellite I DNA competed efficiently with a tRNA gene for binding of yeast RNA polymerase III transcription factors. DNase I footprinting further showed that transcription factor TF IIIC alone bound to satellite I DNA at both the A block and B block consensus promoter sequences immediately downstream from the transcription start point. The strength and position of these associations indicate that satellite I DNA is a potential site for association of the same DNA-binding proteins that activate tRNA gene transcription.

Animals↗
PubMed1986

Processing of mammalian tRNA transcripts in vitro: different pre-tRNAs are processed along alternative pathways that contain a common rate-limiting step.

We have analyzed the pathways and kinetics of processing of mouse tRNA gene transcripts in vitro. Different transcripts are processed along two alternative pathways. The 3' trailer sequence of the tRNA His primary transcript is excised before the 5' leader sequence. In contrast, for the tRNA Gly primary transcript, the 5' leader sequence is excised before the 3' trailer sequence, as has been found for other monomeric eukaryotic tRNA gene transcripts. Computerized analysis of the kinetics of processing indicates that tRNA Asp, tRNA Gly, tRNA Glu and tRNA His transcripts are processed in a substrate concentration-dependent manner and also reveals the existence of a common rate-limiting step, the rate constant of which is equivalent for three of the four transcripts tested. The processing of one pre-tRNA transcript can be competitively inhibited by addition of another pre-tRNA transcript to the processing reaction. The common rate-limiting step is associated with the conversion of the primary transcript to an intermediate and is independent of sequence and the particular processing pathway of the transcript.

Animals↗
PubMed1984

Inhibition of eukaryotic tRNA transcription by potential Z-DNA sequences.

The effect of d(CA/TG)n DNA segments on tRNA transcription has been examined. Alternating purine-pyrimidine tracts were cloned at a long distance from, adjacent to, or within the coding sequence of a tRNAPro gene from Caenorhabditis elegans and shown to be able to assume the A-DNA conformation in vitro in physiological salt concentrations. The transcriptional level of these constructs was compared to that of normal tDNAPro by micro-injection into Xenopus laevis oocytes. Our results show a strong inhibitory effect by potential Z-DNA sequences only when these are placed in the flanking regions of the gene or when they are located between the elements (Box A and Box B) of the split promoter. Transcription was studied in parallel with supercoiled and linear DNA molecules carrying a d(CA/TG) stretch 124-bp long in front of the tRNAPro gene. The results show the same level of inhibition of Po/III transcription regardless of the topological status of the injected DNA.

Animals↗
PubMed1997

Structure and aminoacylation capacities of tRNA transcripts containing deoxyribonucleotides.

The contribution of the ribose 2'-hydroxyls to RNA structure and function has been analyzed, but still remains controversial. In this work, we report the use of a mutant T7 RNA polymerase as a tool in RNA studies, applied to the aspartate and methionine tRNA aminoacylation systems from yeast. Our approach consists of determining the effect of substituting natural ribonucleotides by deoxyribonucleotides in RNA and, thereby, defining the subset of important 2'-hydroxyl groups. We show that deoxyribose-containing RNA can be folded in a global conformation similar to that of natural RNA. Melting curves of tRNAs, obtained by temperature-gradient gel electrophoresis, indicate that in deoxyribo-containing molecules, the thermal stability of the tertiary network drops down, whereas the stability of the secondary structure remains unaltered. Nuclease footprinting reveals a significant increase in the accessibility of both single- and double-stranded regions. As to the functionality of the deoxyribose-containing tRNAs, their in vitro aminoacylation efficiency indicates striking differential effects depending upon the nature of the substituted ribonucleotides. Strongest decrease in charging occurs for yeast initiator tRNA(Met) transcripts containing dG or dC residues and for yeast tRNA(Asp) transcripts with dU or dG. In the aspartate system, the decreased aminoacylation capacities can be correlated with the substitution of the ribose moieties of U11 and G27, disrupting two hydrogen bond contacts with the synthetase. Altogether, this suggests that specific 2'-hydroxyl groups in tRNAs can act as determinants specifying aminoacylation identity.

Base Sequence↗
PubMed1991

Interaction of Escherichia coli tRNA(Ser) with its cognate aminoacyl-tRNA synthetase as determined by footprinting with phosphorothioate-containing tRNA transcripts.

A footprinting technique using phosphorothioate-containing RNA transcripts has been developed and applied to identify contacts between Escherichia coli tRNA(Ser) and its cognate aminoacyl-tRNA synthetase. The cloned gene for the tRNA was transcribed in four reactions in which a different NTP was complemented by 5% of the corresponding nucleoside 5'-O-(1-thiotriphosphate). The phosphorothioate groups of such transcripts are cleaved by reaction with iodine to permit sequencing of the transcripts. Footprinting was achieved by performing the same reaction with the phosphorothioate-tRNA-enzyme complex. At 1 mM iodine, selective protection of the tRNA transcripts in the cognate system was observed, with strong protection at positions 52 and 68 and weak protection at positions 46, 53, 67, 69, and 70. It is suggested that these regions of the tRNA interact with the helical arm of the synthetase.

Base Sequence↗
PubMed1979

The use of rifampicin to evaluate tRNA transcriptional organization in Escherichia coli.

The antibiotic rifampicin, which in prokaryotes inhibits the initiation of RNA synthesis but not the completion of nascent strands, was used to explore tRNA gene transcriptional organization in Escherichia coli. Cultures were grown in [32P] orthophosphate to constant specific radioactivity and labeled with [3H] uridine in the presence of rifampicin. Numerous tRNA species then were isolated by polyacrylamide gel electrophoresis and their 3H/32P ratios determined; these ratios, following correction for the base compositions of the tRNAs, should reflect the distances of the corresponding tRNA genes from their promoters. Individual tRNA species were identified, where possible, by oligonucleotide fingerprint analysis. Observed isotopic ratios were correlated with promoter-gene distances, measured in nucleotides, using the nucleotide sequence of the 16S ribosomal RNA gene as a reference. The protocols developed should be applicable to most prokaryotes.

Base Sequence↗
PubMed1993

Synthetic RNA-cleaving molecules mimicking ribonuclease A active center. Design and cleavage of tRNA transcripts.

RNA cleaving molecules were synthesized by conjugating imidazole residues imitating the essential imidazoles in the active center of pancreatic ribonuclease to an intercalating compound, derivative of phenazine capable of binding to the double stranded regions of polynucleotides. Action of the molecules on tRNA was investigated. It was found, that some of the compounds bearing two imidazole residues cleave tRNA under physiological conditions. The cleavage reaction shows a bell-shaped pH dependence with a maximum at pH 7.0 indicating participation of protonated and non-protonated imidazole residues in the process. Under the conditions stabilizing the tRNA structure, a tRNAAsp transcript was cleaved preferentially at the junctions of the stem and loop regions of the cloverleaf tRNA fold, at the five positions C56, C43, C20.1, U13, and U8, with a marked preference for C56. This cleavage pattern is consistent with a hydrolysis mechanism involving non-covalent binding of the compounds to the double-stranded regions of tRNA followed by an attack of the imidazole residues at the juxtaposed flexible single-stranded regions of the molecule. The compounds provide new probes for the investigation of RNA structure in solution and potential reactive groups for antisense oligonucleotide derivatives.

Base Sequence↗
PubMed1980

Transcription of tRNA genes in vivo: single-stranded compared to double-stranded templates.

The expression of cloned tRNA genes has been studied by injecting single-stranded and double-stranded DNA templates into Xenopus oocyte nuclei. In both forms the genes are faithfully transcribed after injection. Some single-stranded DNA is converted into double-stranded DNA in the oocyte nucleus. This conversion is necessary for the expression of the injected tRNA gene: no tRNA transcription is observed when DNA synthesis is inhibited. We conclude that single-stranded DNA does not serve as a template for faithful transcription of this gene in injected oocytes.

Coliphages↗
PubMed1989

Multiple mutations of the first gene of a dimeric tRNA gene abolish in vitro tRNA gene transcription.

Eukaryotic tRNA expression initiates with transcription by RNA polymerase III and requires two additional protein factors and two regions within the tRNA gene (the 5'-internal control region (ICR) or A-box and the 3'-ICR or B-box). Using a reconstituted Saccharomyces cerevisiae RNA polymerase III system, the transcription of various 5'-ICR, 3'-ICR, and double mutation alleles of the Schizosaccharomyces pombe sup3-e dimeric tRNA gene were studied. The sup3-e tRNA locus consists of an upstream serine tRNA gene and a downstream initiator methionine tRNA gene which are transcribed as a dimeric precursor and processed to give two tRNAs. Only the ICRs of the tRNA(Ser) gene are active in directing dimeric gene transcription. Mutations in the 3'-ICR of the tRNA(Ser) gene reduce transcription of the dimer more than those in the 5'-ICR. Mutations in the 5'-ICR were found which greatly increased or decreased transcription of the dimer, while base changes in the 3'-ICR were only found to decrease transcription. This suggests a modulatory role for the 5'-ICR in transcription regulation. Mutation of the methionine tRNA gene ICR has little effect on sup3-e transcription, and no detectable transcripts initiate from the methionine tRNA gene when the tRNA(Ser) gene promoter is inactivated by mutation. Comparison with transcription studies of other mutant tRNA genes suggests that nucleotides sites within the ICRs, such as nucleotides 8, 10, 13, 18, and 19 in the 5'-ICR and 48, 53, 56, 57, and 58 in the 3'-ICR, appear to have evolved universal importance for RNA polymerase III transcription in eukaryotes. Thus these ICR sequences may play a critical role in regulation of tRNA expression.

Cloning, Molecular↗
PubMed1991

Analysis of the molecular mechanisms for the species-specific transcription of Drosophila and human tRNA gene transcription components.

The transcription of eucaryotic tRNA genes requires two factors IIIB and IIIC, in addition to RNA polymerase III, to reconstitute this process in vitro. We have examined the functional exchangeability of these components from Drosophila and human systems. The reconstitution of heterologous IIIB and IIIC components demonstrated that neither factor will functionally substitute for the homologous components to activate tRNA gene transcription. The addition of the heterologous Drosophila factors to HeLa transcription assays causes an inhibition of RNA synthesis that is dependent upon the order of addition of these proteins to the DNA template. Thus, it appears that tRNA gene transcription in these systems is species-specific. We have further analyzed the reason for the apparent incompatibilities of these components by the use of stable complex formation assays. We find that human HeLa IIIB and Drosophila IIIC are unable to form stably associated complexes with a tRNA gene template, whereas the Drosophila IIIB and HeLa IIIC do form stable but nonproductive complexes. These results demonstrate that specific IIIC-IIIB interactions are critical in the formation of productive transcription complexes and are responsible for the observed species specificity of Drosophila and human tRNA gene transcription.

Animals↗
PubMed2001

Identity elements in tRNA-mediated transcription antitermination: implication of tRNA D- and T-arms in mRNA recognition.

tRNA-mediated transcription antitermination has been shown to control the expression of several amino acid biosynthesis operons and aminoacyl-tRNA-synthetase-encoding genes in Gram-positive bacteria. A model originally put forward by Grundy & Henkin describes the conserved structural features of the leader sequences of these operons and genes. Two sequences of 3 and 4 nt, respectively, take a central position in this model and are thought to be responsible for the binding of the system-specific uncharged tRNA, an interaction which would stabilize the antiterminator conformation of the leader. Here a further evolution of this model is presented based on an analysis of trp regulation in Lactococcus lactis in which a function is assigned to hitherto unexplained conserved structures in the leader sequence. It is postulated that the mRNA-tRNA interaction involves various parts of the tRNA in addition to the anticodon and the acceptor in the original model and that these additional interactions contribute to the recognition of a specific tRNA, and hence to the specificity and efficacy of the regulatory response.

5' Untranslated Regions↗
PubMed1984

Level of rRNA, not tRNA, synthesis controls transcription of rRNA and tRNA operons in Escherichia coli.

We have recently proposed a model for the negative feedback control of rRNA and tRNA synthesis in Escherichia coli by products of rRNA operons or their derivatives (e.g., nontranslating ribosomes) (S. Jinks-Robertson, R.L. Gourse, and M. Nomura, Cell 33:865-876, 1983). In this paper, we examined the following questions. (i) Are the spacer tRNAs carried within rRNA operons the products responsible for the regulation of rRNA and tRNA transcription? (ii) Are tRNAs capable of regulating their own syntheses? We measured tRNA accumulations in cells containing plasmids with intact or defective rRNA operons or with tRNA operons. From the results obtained, we conclude that neither the tRNAs encoded within rRNA operons nor the tRNAs encoded in non-rRNA operons are capable of controlling rRNA or tRNA transcription. Therefore, the products responsible for the initial step leading to rRNA and tRNA regulation are rRNAs (or their derivatives).

Aspartate-tRNA Ligase↗
PubMed1985

A large region controls tRNA gene transcription.

We find that tRNA gene transcription is controlled by a region much larger than the previously described internal segments known as A and B boxes. In a Bombyx mori silkworm tRNA2Ala gene, transcriptionally important sequences extend at least from -13 to +146, and thus include sequences both upstream and downstream from the 98 base-pair primary transcription unit. We show that the apparent size of this control region can be manipulated by the conditions used to measure transcription in vitro. Specifically, the use of high concentrations of templates to overcome the effect of an inhibitory substance in crude extracts can make the control region appear smaller. We propose that this finding explains much of the observed variability in the sequence requirements for transcription of different tRNA genes. If so, large size is likely to be a general feature of tRNA gene control regions.

Animals↗
PubMed1982

The minimum intragenic sequences required for promotion of eukaryotic tRNA gene transcription.

Transcription of eukaryotic tRNA genes is controlled by two intragenic regions, the D-control region (which in the tRNA codes for the D-stem and -loop) and the T-control region (which in the tRNA codes for the T psi C loop). To determine whether these sequences alone are sufficient to promote tRNA gene transcription in vitro, the two control regions of a Drosophila tRNAArg gene were cloned separately from the context of the parental DNA (these constructions are called tRNA minigenes). The tRNA minigene that contains both intragenic control regions supports in vitro RNA synthesis in Xenopus laevis oocyte and HeLa cell transcription systems. The mutant which has deletions to nucleotide 7 within the mature tRNA coding region, pArg5.7, and minigenes derived from it do not support RNA synthesis in a Drosophila Kc cell transcription system. Xenopus and Hela extracts transcribe pArg5.7 albeit at reduced levels compared to the wild-type gene. The tRNA minigene that contained only the D-control region was not able to support RNA synthesis in any of these three transcription systems. A mutant tRNA gene comprising the 3' half of the tRNAArg gene similarly was not able to support RNA synthesis. These experiments show that the DNA sequence from nucleotides 7-58, which contains both intragenic control regions of the tRNA gene, possesses sufficient information to initiate specific transcription by RNA polymerase III in Xenopus and HeLa systems. The transcription efficiency of this tRNA minigene however is reduced to about 20% the transcription level of the wild type tRNA gene. This lowered level of transcriptional efficiency results from deleting the ends of the native tRNA gene and its adjacent flanking sequences. The affects of deleting 5' sequences are most pronounced in the Drosophila transcription system.

Animals↗