Interpretation of incomplete reactions in tRNA aminoacylation. Aminoacylation of yeast tRNA Val II with yeast valyl-tRNA synthetase.
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Adenosine Monophosphate↗
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Large scale purification of tRNA ser , tRNA tyr and tRNA phe from Baker's yeast.
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Acylation↗
Generality of the tRNA origin of short interspersed repetitive elements (SINEs). Characterization of three different tRNA-derived retroposons in the octopus.
Transcription in vitro of total genomic DNA of Octopus vulgaris resulted in PolIII transcripts of 200 to 500 nucleotides in length. These transcripts were used as probes and, as a result, three different kinds of short interspersed element (SINE) were isolated and characterized. Two SINEs, designated the octopus OR1 and OR2 families, seem to have been derived from tRNA(Arg). The other SINE, designated the octopus OK family, have originated from tRNA, but the parental tRNA species cannot be identified due to sequence divergence from the original tRNA sequence. The OR1 and OR2 families exhibit considerable similarity to one another, in the 5' region of the tRNA-unrelated region as well as in the tRNA(Arg)-related region, an observation that suggests that these two families may have had the same origin in evolution. The three SINEs together constitute at least 6% of the genome of O. vulgaris. Results of a dot hybridization experiment suggest that the OR1 and OR2 families are present in fewer species than the OK family and that the OK family exists in many species of octopodid. The present observations indicate that SINEs have generally been derived from tRNAs in invertebrates, as well as in vertebrates, and that retroposition is widely involved in the genetic and structural variability of invertebrate genomes. It has been demonstrated that tRNA(Arg) and tRNA(Lys) appear to be tRNA species found as progenitors of vertebrate SINEs, and the same preference for progenitor species of tRNAs seems also to be a feature of invertebrate SINEs.
Animals↗
RNA editing of tRNA(Phe) and tRNA(Cys) in mitochondria of Oenothera berteriana is initiated in precursor molecules.
We have analyzed the role of RNA editing in the correction of mismatched base pairs in tRNA secondary structures in mitochondria of the flowering plant Oenothera berteriana. Comparison of genomic and cDNA sequences from unprocessed primary transcripts of the newly characterized genes for tRNA(Cys), tRNA(Asn) and tRNA(Ile) and the previously described gene for tRNA(Phe) revealed single nucleotide discrepancies in the tRNA(Cys) and tRNA(Phe) sequences. While the change in the anticodon stem of tRNA(Cys) alters a C-T to a T-T mismatch, the nucleotide transition in the tRNA(Phe) restores a conventional T-A Watson-Crick base pair, replacing a C-A mismatch in the acceptor stem. Since both nucleotide alterations are conversions from genomic cytidines to thymidines in the cDNA (uridines in the tRNAs), they are attributed to RNA editing, which is observed in nearly all mRNAs from plant mitochondria.
Base Sequence↗
A new gene encoding tRNA(Pro) (GGG) is present in the chloroplast genome of black pine: a compilation of 32 tRNA genes from black pine chloroplasts.
The chloroplast genome of black pine (Pinus thunbergii), a gymnosperm, contains 32 different tRNA genes, 30 of which correspond to those previously identified in tobacco and rice chloroplast genomes. Two additional genes encode tRNA(Pro) (GGG) and tRNA(Arg) (CCG); the former is newly identified while the latter is present in liverwort, Physcomitrella patens and Angiopteris lygodiifolia, chloroplast genomes. Moreover, a partial copy of the split tRNA(Gly) (UCC) gene and full copies of tRNA(His) (GUG), tRNA(Thr) (GGU) and tRNA(Ser) (GCU) genes are present in the large single-copy region of the genome, suggesting extensive rearrangements of the chloroplast genome during evolution. No tRNA genes whose tRNA products can recognize codons CUU/C (Leu) and GCU/C (Ala) have been found. We propose that the 32 tRNAs are sufficient to read all the 61 sense codons in the black pine system using the "two-out-of-three" and the "U:N wobble" mechanisms.
Base Sequence↗
Organization and structure of an E. coli tRNA operon containing seven tRNA genes.
The structure and organization on the Escherichia coli chromosome of the gene cluster coding for two methionine tRNAs (tRNAmMet), four glutamine tRNAs (two tRNA1Gln and two tRNA2Gln), and a previously unidentified tRNA (called tRNAx) have been studied by restriction enzyme analysis and DNA sequencing, utilizing a specialized transducing bacteriophage (lambda psu degrees 2) carrying the supB-supE region. From the sequence analysis, the previously unidentified tRNA has been shown to have an anticodon sequence (5'-UAG-3') corresponding to a leucine codon. The organization of this tRNA gene cluster on the E. coli chromosome is tRNAmMet-9 base pairs-tRNAx-23 base pairs-tRNA1Gln-34 base pairs-tRNA1Gln-15 base pairs-tRNAmMet-47 base pairs-tRNA2Gln-37 base pairs-tRNA2Gln. The duplicated genes coding for tRNAmMet, tRNA,Gln, and tRNA2Gln have identical sequences, which are the same as the sequences determined previously with tRNA molecules. These tRNA sequences are preceded by a single promoter region where a "Pribnow box" sequence is present seven base pairs upstream from the transcription start site. The spacer regions separating the seven tRNA sequences are different from each other both in size and in nucleotide sequence. The possible implication of these sequences for precursor processing is discussed. A restriction fragment that has been originally identified in lambda psu degrees 2 DNA and shown to contain the seven tRNA genes has been detected in the E. coli chromosome, thereby suggesting that this tRNA gene cluster is present in the bacterial genome with the same organization as in the transducing phage genome.
Base Sequence↗
tRNA-guanine transglycosylase from Escherichia coli: recognition of dimeric, unmodified tRNA(Tyr).
In order to probe the interaction between tRNA and the tRNA hypermodifying enzyme, tRNA-guanine transglycosylase (TGT) from Escherichia coli, we have undertaken the generation of E coli tRNA(Tyr) and analogues. During efforts to adapt currently available in vitro transcription techniques we encountered difficulties attributable to dimerization of the tRNA products. E coli tRNA(Tyr) has previously been characterized for its ability to form a dimer in solutions of suitable salt concentrations at appropriate temperatures (Yang SK, Söll DG, Crothers DM (1972) Biochemistry 11, 2311-2320; Rordorff BF, Kearns DR (1976) Biochemistry 15, 3320-3330). We have applied similar techniques to our unmodified analogue of E coli tRNA(Tyr) and produced both monomeric and dimeric forms of E coli tRNA(Tyr). In this report we find that the dimer does serve as a substrate for modification by TGT. While both the conformers are equal in terms of Vmax (within experimental error) a 2.5-fold increase in KM occurs when going from monomer to dimer. This suggests that TGT preferentially binds the monomer but once either conformer is bound will catalyze the modification reaction equally well. We have also compared the results for the two conformers to our previous data of an RNA minihelix corresponding to the anticodon arm of E coli tRNA(Tyr). Here we find that our earlier conclusion, that the recognition elements for TGT are localized within the anticodon arm of cognate tRNAs, is supported.
Base Sequence↗
Suppressor mutations in Escherichia coli methionyl-tRNA formyltransferase that compensate for the formylation defect of a mutant tRNA aminoacylated with lysine.
The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in eubacteria such as Escherichia coli. In addition to the determinants for formylation present in the initiator tRNA, the nature of the amino acid attached to the tRNA is also important for formylation. We showed previously that a mutant tRNA aminoacylated with lysine was an extremely poor substrate for formylation. As a consequence, it was essentially inactive in initiation of protein synthesis in E. coli. In contrast, the same tRNA, when aminoacylated with methionine, was a good substrate for formylation and was, consequently, quite active in initiation. Here, we report on the isolation of suppressor mutations in MTF which compensate for the formylation defect of the mutant tRNA aminoacylated with lysine. The suppressor mutant has glycine 178 changed to glutamic acid. Mutants with glycine 178 of MTF changed to aspartic acid, lysine, and leucine were generated and were found to be progressively weaker suppressors. Studies on allele specificity of suppression using different mutant tRNAs as substrates suggest that the Gly178 to Glu mutation compensates for the nature of the amino acid attached to the tRNA. We discuss these results in the framework of the crystal structure of the MTF.fMet-tRNA complex published recently.
Acylation↗
Isolation of Saccharomyces cerevisiae mitochondrial formyltetrahydrofolic acid:methionyl-tRNA transformylase and the hybridization of mitochondrial fMet-tRNA with mitochondrial DNA.
Formyltetrahydrofolic acid:methionyl-tRNA transformylase was isolated from Saccharomyces cerevisiae mitochondria and used to prepare yeast mitochondrial [(3)H]formylmethionyl-tRNA. This fMet-tRNA hybridizes with mitochondrial DNA but not with yeast nuclear or E. coli DNA. Unlabeled mitochondrial, but not extramitochondrial, tRNA competes in this reaction. tRNA was eluted from the hybrid and found to contain the label almost exclusively in fMet-tRNA. Yeast cytoplasmic fMet-tRNA formylated with Escherichia coli enzyme, and E. coli fMet-tRNA, do not hybridize with mitochondrial DNA. It is concluded that yeast mitochondrial tRNA(fMet) is a gene product of the mitochondrial genome.
DNA↗
NMR analysis of tRNA acceptor stem microhelices: discriminator base change affects tRNA conformation at the 3' end.
An important step in initiation of protein synthesis in Escherichia coli is the specific formylation of the initiator methionyl-tRNA (Met-tRNA) by Met-tRNA transformylase. The determinants for formylation are clustered mostly in the acceptor stem of the initiator tRNA. Here we use NMR spectroscopy to characterize the conformation of two RNA microhelices, which correspond to the acceptor stem of mutants of E. coli initiator tRNA and which differ only at the position corresponding to the "discriminator base" in tRNAs. One of the mutant tRNAs is an extremely poor substrate for Met-tRNA transformylase, whereas the other one is a much better substrate. We show that one microhelix forms a structure in which its 3'-ACCA sequence extends the stacking of the acceptor stem. The other microhelix forms a structure in which its 3'-UCCA sequence folds back such that the 3'-terminal A22 is in close proximity to G1. These results highlight the importance of the discriminator base in determining tRNA conformation at the 3' end. They also suggest a correlation between tRNA structure at the 3' end and its recognition by Met-tRNA transformylase.
Base Sequence↗
lambda bar minigene-mediated inhibition of protein synthesis involves accumulation of peptidyl-tRNA and starvation for tRNA.
Expression of the bacteriophage lambda two-codon, AUG AUA, barI minigene (bar+) leads to the arrest of protein synthesis in cells defective in peptidyl-tRNA hydrolase (Pth). It has been hypothesized that translation of the bar+ transcript provokes premature release and accumulation of peptidyl-tRNA (p-tRNA). Inhibition of protein synthesis would then result from either starvation of sequestered tRNA or from toxicity of accumulated p-tRNA. To test this hypothesis and to investigate the cause of arrest, we used a coupled in vitro transcription-translation system primed with DNA containing bar+ and the beta-lactamase-encoding gene of the vector as a reporter. The results show that expression of bar+ minigene severely inhibits beta-lactamase polypeptide synthesis by Pth-defective extracts and partially inhibits synthesis by wild-type extracts. Fractions enriched for Pth, or a homogeneous preparation of Pth, prevented and reversed bar+-mediated inhibition. A mutant minigene, barA702, which changes the second codon AUA (Ile) to AAA (Lys), was also toxic for Pth-defective cells. Expression of barA702 inhibited in vitro polypeptide synthesis by Pth-defective extracts and, as with bar+, exogenous Pth prevented inhibition. Addition of pure tRNALys prevented inhibition by barA702 but not by bar+. Expression of bar+ and barA702 led to release and accumulation of p-tRNAIle and p-tRNALys respectively but bar+ also induced accumulation of p-tRNALys. Finally, bar+ stimulated association of methionine with ribosomes probably as fMet-tRNAfMet and the accumulation of methionine and isoleucine in solution as peptidyl-tRNA (p-tRNA). These results indicate that minigene-mediated inhibition of protein synthesis involves premature release of p-tRNA, misincorporation of amino acyl-tRNA, accumulation of p-tRNAs and possibly sequestration of tRNAs.
Bacteriophage lambda↗
Pyrophosphate mediates the effect of certain tRNA mutations on aminoacylation of yeast tRNA(Phe).
The influence of pyrophosphate hydrolysis by inorganic pyrophosphatase on homologous aminoacylation of different yeast tRNA(Phe) mutants was studied. The addition of pyrophosphatase significantly improved the aminoacylation efficiency of tRNA(Phe) structural mutants as well as the mutant with substitution at position 20, while having no effect on the charge of wild-type tRNA(Phe). Aminoacylation of tRNA(Phe) anticodon and discriminator base (N(73)) mutants was not affected by pyrophosphatase. Activation of wild-type tRNA(Phe) transcript aminoacylation by inorganic pyrophosphatase was observed only at low Mg(2+) concentrations due to distortion of the tRNA(Phe) structure under these conditions. Our results demonstrate that pyrophosphate dissociation becomes a rate-limiting step of the reaction in yeast phenylalanyl-tRNA synthetase catalyzed aminoacylation of tRNA(Phe) variants with altered tertiary structure. A possible mechanism of pyrophosphate-mediated inhibition of tRNA mutants aminoacylation is discussed.
Acylation↗
Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication.
Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNA(Pro)molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian retro-viruses have revealed evidence of molecular adapt-ation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites. While most of the selected primer binding sites are complementary to the 3'-end of tRNA((Pro)), we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNA(Arg(CCU)), tRNA(Phe(GAA))and a hitherto unknown human tRNA(Ser(CGA)).
3T3 Cells↗
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↗
Compilation of tRNA sequences and sequences of tRNA genes.
Maintained at the Universitat Bayreuth, Bayreuth, Germany, the Compilation of tRNA Sequences and Sequences of tRNA Genes is accessible at the URL http://www.tRNA.uni-bayreuth.de with mirror site located at the Institute of Protein Research, Pushchino, Russia (http://alpha.protres.ru/trnadbase). The compilation is a searchable, periodically updated database of currently available tRNA sequences. The present version of the database contains a new Genomic tRNA Compilation including the sequences of tRNA genes from genomic sequences published up to July 2003. It consists of about 5800 tRNA gene sequences from 111 organisms covering archaea, bacteria, higher and lower eukarya. The former Compilation of tRNA Genes (up to the end of 1998) and the updated Compilation tRNA Sequences (561 entries) are also supported by the new software. The database can be explored by using multiple search criteria and sequence templates. The database provides a service that allows to obtain statistical information on the occurrences of certain bases at given positions of the tRNA sequences. This allows phylogenic studies and search for identity elements in respect to interactions of tRNAs with various enzymes.
Animals↗
Proton exchange and basepair kinetics of yeast tRNA(Phe) and tRNA(Asp1).
Nuclear magnetic resonance measurements of proton exchange were performed on yeast tRNA(Phe) and yeast tRNA(Asp), at temperatures from 20 to 45 degrees C, in the presence of various levels of salt, phosphate, added magnesium, and pH. The dynamical changes of the tRNA molecule were interpreted, with the aid of firmly established assignments and the use of the saturation recovery technique. In tRNA(Phe), the exchange rates in zero magnesium indicated early melting of the acceptor stem, tertiary structure, and D stem. However, in the presence of even low levels of magnesium the D stem remained intact up to high temperature, stabilized by a Mg2+ ion. A similar unfolding sequence was observed in tRNA(Asp). The difference between these two tRNAs was the thermal behavior of the tertiary resonance U8-A14. In tRNA(Phe), this base pair showed sharp rate increases between 32 and 39 degrees C. However, in tRNA(Asp), it remained intact up to 36 degrees C and disappeared at 39 degrees C, even if there was not important kinetic broadening. By measuring the temperature dependence of the exchange rates, we obtained an activation energy of 40-60 kcal/mol for all the imino protons of yeast tRNA(Phe) in zero magnesium. The same activation energy was obtained for tRNA(Phe) with equimolar concentration of magnesium. By investigation of the dependence of the exchange rates of these imino protons on solution conditions, we observed the transition from kop rate limiting in the absence of magnesium to kex rate limiting in the presence of magnesium.
Base Composition↗
Differential synthesis rates of tRNA species in the silk gland of Bombyx mori are required to promote tRNA adaptation to silk messages.
In studying the mechanism of tRNA adaptation to silk fibroin and sericin mRNA translation, we investigated the accumulation rates of tRNA species rapidly labeled in vivo in the posterior and middle silk glands of the silkworm Bombyx mori during the last larval instar. We found that the rates of synthesis of total and individual tRNA species labeled with [3H]uridine during the growth and the secretion phases and separated by electrophoresis on polyacrylamide gels, are specific for each tRNA species and each tissue. We noted continuous variations of the tRNA levels designed to reflect changes in the population of mRNAs. The tRNA adaptation to silk mRNAs is completed roughly at the end of the growth phase and at the beginning of the secretion phase (day four of the Vth instar). Its stabilization during the secretion phase occurs by a reversal of the relative rates of predominant tRNA species, mainly tRNAGly. The sum total of the synthetic rates for each tRNA species corroborates their known adaptive distribution during the secretion phase of silk proteins. In addition, when coupled to the similarity of their turnover rates, observed previously, the data support a transcriptional control of tRNA genes.
Animals↗
Expression in Escherichia coli of Bacillus subtilis tRNA genes from a promoter within the tRNA gene region.
A cloned DNA segment from Bacillus subtilis containing 21 tRNA genes was introduced into Escherichia coli. In the B. subtilis genome, these tRNA genes are located after an rRNA gene set and before tandem terminators. The rRNA and tRNA genes are thought to represent a single transcriptional unit. However, another putative promoter occurs after the second tRNA gene within the tRNA gene cluster and has a sequence compatible with both the major B. subtilis (sigma 43 type) promoter and the major E. coli promoter. The B. subtilis 21-tRNA-gene cluster was introduced into E. coli to see whether this promoter would be recognized in E. coli, to determine the start point of transcription in the E. coli system, and to see whether mature B. subtilis tRNAs would be transcribed and processed in E. coli. Expression was evaluated by monitoring levels of aminoacylation of mature tRNAs extracted from E. coli containing plasmids with or without the B. subtilis tRNA genes and by examining profiles of isoaccepting species on columns of RPC-5. S1 nuclease mapping was performed to define the starting point for transcription. The results indicated that a putative promoter located within the B. subtilis tRNA gene region was functional when cloned into E. coli and that it initiated at the same nucleotide as it does in B. subtilis. In addition, at least some B. subtilis tRNA genes could be transcribed and processed in E. coli to mature tRNAs capable of accepting an amino acid.
Bacillus subtilis↗