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PubMed1988

Discrimination between glutaminyl-tRNA synthetase and seryl-tRNA synthetase involves nucleotides in the acceptor helix of tRNA.

Analysis of the in vivo amber suppressor activity of mutants derived from two Escherichia coli serine tRNAs shows that substitution of 2 base pairs in the acceptor helix changes a serine suppressor tRNA to an efficient glutamine acceptor. Determination of the amino acid inserted in vivo into protein by this tRNA shows that these changes reduce the tRNA recognition by seryl-tRNA synthetase while increasing that of glutaminyl-tRNA synthetase. This implies that misaminoacylation in vivo is dependent on the competition by different synthetases for the tRNA. In addition, the "translational efficiency" of tRNA is an integral part in observing misaminoacylation in vivo.

Amino Acyl-tRNA Synthetases↗
PubMed2000

Arc1p organizes the yeast aminoacyl-tRNA synthetase complex and stabilizes its interaction with the cognate tRNAs.

Eukaryotic aminoacyl-tRNA synthetases, in contrast to their prokaryotic counterparts, are often part of high molecular weight complexes. In yeast, two enzymes, the methionyl- and glutamyl-tRNA synthetases associate in vivo with the tRNA-binding protein Arc1p. To study the assembly and function of this complex, we have reconstituted it in vitro from individually purified recombinant proteins. Our results show that Arc1p can readily bind to either or both of the two enzymes, mediating the formation of the respective binary or ternary complexes. Under competition conditions, Arc1p alone exhibits broad specificity and interacts with a defined set of tRNA species. Nevertheless, the in vitro reconstituted Arc1p-containing enzyme complexes can bind only to their cognate tRNAs and tighter than the corresponding monomeric enzymes. These results demonstrate that the organization of aminoacyl-tRNA synthetases with general tRNA-binding proteins into multimeric complexes can stimulate their catalytic efficiency and, therefore, offer a significant advantage to the eukaryotic cell.

Catalysis↗
PubMed1990

Exchange of spacer regions between rRNA operons in Escherichia coli.

The Escherichia coli rRNA operons each have one of two types of spacer separating the 16S and 23S coding regions. The spacers of four operons encode tRNA(Glu2) and the other three encode both tRNA(Ile) and tRNA(Ala1B). We have prepared a series of mutants in which the spacer region of a particular rrn operon has been replaced by the opposite type. Included among these were a mutant retaining only a single copy of the tRNA(Glu2) spacer (at rrnG) and another retaining only a single copy of the tRNA(Ile)-tRNA(Ala1B) spacer (at rrnA). While both mutants grew more slowly than controls, the mutant deficient in tRNA(Glu2) spacers was more severely affected. At a frequency of 6 X 10(-5), these mutants phenotypically reverted to faster growing types by increasing the copy number of the deficient spacer. In most of these phenotypic revertants, the deficient spacer type appeared in a rrn operon which previously contained the surplus type, bringing the ratio of spacer types closer to normal. In a few cases, these spacer changes were accompanied by an inversion of the chromosomal material between the donor and recipient rrn operons. Two examples of inversion of one-half of the E. coli chromosome between rrnG and rrnH were observed. The correlation of spacer change with inversion indicated that, in these particular cases, the change was due to an intrachromatid gene conversion event accompanied by a reciprocal crossover rather than reciprocal exchange between sister chromatids.

Base Sequence↗
PubMed1989

Three mitochondrial tRNA genes from Arabidopsis thaliana: evidence for the conversion of a tRNAPhe gene into a tRNATyr gene.

Three tRNA genes have been isolated from a genomic library of Arabidopsis thaliana: a tRNASer (GCU), a tRNATyr (GUA) and a tRNAGlu (UUC) genes. These genes are located closely on the same DNA fragment. The tRNASer and the tRNAGlu genes have both 99% sequence similarity with their mitochondrial counterparts from higher plants indicating that these three tRNA genes are mitochondrial. The tRNATyr gene shows a particular high sequence similarity with the mitochondrial tRNAPhe pseudogene from maize, and both genes are flanked by a tRNASer gene in the upstream region. Extensive sequence comparisons of the Arabidopsis thaliana mitochondrial sequence containing the three tRNA genes and the corresponding region from maize and soybean mitochondria have shown evidence that the tRNA Tyr gene has been generated from a mitochondrial tRNAPhe gene. The conversion was accomplished by three genetic events: a 4 base-pair deletion, a mutation and a recombination, which led to the transformation of the acceptor stem and the anticodon.

Base Sequence↗
PubMed1994

Contacts between 16S ribosomal RNA and mRNA, within the spacer region separating the AUG initiator codon and the Shine-Dalgarno sequence; a site-directed cross-linking study.

mRNA analogues containing several 4-thiouridine (thio-U) residues at selected positions were prepared by T7-transcription. The spacer region between the Shine-Dalgarno sequence and the AUG codon consisted of four or eight bases with a single thio-U at a variable position; alternatively, cro-mRNA analogues were used carrying the thio-U substituted spacer sequence UUGU. The mRNAs were bound to E. coli ribosomes, and--after irradiation--the sites of cross-linking to 16S RNA were analysed. Three cross-links to the 16S RNA from the spacer region were observed, namely to positions 665, 1360, and a site close to nucleotide 1530. The cross-links were formed in different amounts in the presence or absence of tRNA(fMet), and were observed from thio-U residues located at various positions within the spacer sequence, although in the presence of tRNA they were in general stronger from positions close to the Shine-Dalgarno end of the spacer. The cross-linking behaviour in this upstream area of the mRNA is thus rather different in character from the previously published pattern in the downstream area. From considerations of structural conservation in small subunit RNA, we propose that both the upstream and downstream cross-links to 16S RNA reflect a universal mRNA path through the ribosome.

Base Sequence↗
PubMed1995

A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity.

We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The non-destructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GlnRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H]Gln-tRNA or [14C]Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyl-tRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GlnRS.

Acylation↗
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↗
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↗
PubMed2003

Nucleolar clustering of dispersed tRNA genes.

Early transfer RNA (tRNA) processing events in Saccharomyces cerevisiae are coordinated in the nucleolus, the site normally associated with ribosome biosynthesis. To test whether spatial organization of the tRNA pathway begins with nucleolar clustering of the genes, we have probed the subnuclear location of five different tRNA gene families. The results show that tRNA genes, though dispersed in the linear genome, colocalize with 5S ribosomal DNA and U14 small nucleolar RNA at the nucleolus. Nucleolar localization requires tRNA gene transcription-complex formation, because inactivation of the promoter at a single locus removes its nucleolar association. This organization of tRNA genes must profoundly affect the spatial packaging of the genome and raises the question of whether gene types might be coordinated in three dimensions to regulate transcription.

Cell Nucleolus↗
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↗
PubMed2004

Mitochondrial 3' tRNA editing in the jakobid Seculamonas ecuadoriensis: a novel mechanism and implications for tRNA processing.

The jakobid flagellates are bacteriovorus protists with mitochondrial genomes that are the most ancestral identified to date, in that they most resemble the genomes of the alpha-proteobacterial ancestors of the mitochondrion. Because of the bacterial character of jakobid mitochondrial genomes, it was expected that mechanisms for gene expression and RNA structures would be bacterial in nature. However, sequencing of the mitochondrial genome of the jakobid Seculamonas ecuadoriensis revealed several apparent mismatches in the acceptor stems of two predicted tRNAs. To investigate this observation, we determined the cDNA sequences of these tRNAs by RT-PCR. Our results show that the last three positions of the 3' extremity, plus the discriminator position of seryl and glutamyl tRNAs, are altered posttranscriptionally, restoring orthodox base-pairing and replacing the discriminator with an adenosine residue, in an editing process that resembles that of the metazoan Lithobius forficatus. However, the most 5' of the edited nucleotides is occasionally left unedited, indicating that the editing mechanism proceeds initially by exonucleolytic degradation, followed by repair of the degraded region. This 3' tRNA editing mechanism is likely distinct from that of L. forficatus, despite the apparent similarities between the two systems.

Animals↗
PubMedDate not supplied

Selective binding of amino acid residues to tRNA molecules detected by anticodon-anticodon interactions.

Anticodon-anticodon pairing of complementary tRNA's has been studied by fluorescence temperature jump measurements in the presence of different ligands as an approach for the evaluation of ligand binding to tRNA. This procedure is particularly useful for ligands which do not show spectroscopic changes upon binding, but affect the pairing potential of anticodons. Addition of phenylalanine-, tyrosine- and tryptophan-amide leads to a substantial decrease of the tRNAPhe.tRNAGlu pairing constant Kp, whereas Kp remains almost unaffected by addition of leucine amide and increases upon addition of glycine amide. The effects observed for the aromatic amino acid amides can be described quantitatively by a site binding model with preferential binding of the amides to tRNAPhe. The binding constants evaluated according to this model (Phe-amide 120 M-1, Tyr-amide 160 M-1 and Trp-amide 580 M-1) are consistent with values obtained independently by fluorescence titrations with tRNAPhe. Selective binding of these amino acid residues to tRNAPhe is deduced from the observed concentration dependence which is not compatible with a corresponding binding process to tRNAGlu. Addition of glutamic acid diamide induces an increase of the tRNAPhe.tRNAGlu pairing constant, which is however equivalent to that observed for tRNAPhe.tRNALys pairing and thus does not demonstrate a selective binding to tRNAGlu. The pairing of tRNAPhe with tRNAGlu is strongly enhanced by addition of Mg2+ or spermine. Evaluation of the Mg2+ data by a site model leads to constants of 360 M-1 for the binding of Mg2+ to monomer tRNA and 3000 M-1 for the binding of Mg2+ to the tRNAPhe.tRNAGlu dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1995

Identification and mapping of trnI, trnE and trnfM genes in the sunflower mitochondrial genome.

Three sunflower mitochondrial HindIII restriction fragments containing the tRNA genes trnI, trnE and trnfM have been sequenced. The genes are present in single copy on the whole genome and are transcribed. Hybridization experiments and sequence analysis of the HindIII fragments allowed the precise mapping and orientation of each gene on the sunflower mitochondrial genome.

Base Sequence↗
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↗
PubMed1990

Temperature sensitive synthesis of transfer RNAs in vivo in Saccharomyces cerevisiae.

Dictyostelium discoideum tRNA genes can be expressed efficiently in vivo in yeast, and transcription products are processed to mature tRNAs. However, primary transcripts of a variant tRNA(Val)(UAC) gene are processing deficient under standard growth conditions (30 degrees C), due to a slightly altered 5' flanking region. A stable extended amino acid acceptor stem, which seems to be required to compensate a G5-G68 mismatch, cannot form. This mismatch destabilizes secondary and probably tertiary structures to such an extent that recognition of processing enzyme(s) under normal conditions (30 degrees C) is impaired. Growing yeast cells at reduced temperature (22 degrees C) can phenotypically complement the processing defect. This observation provides a new concept for the temperature dependent expression of protein coding genes which carry a nonsense codon. Translation of corresponding messages can be controlled by products of a temperature sensitive su-tRNA gene. We successfully tested this concept with two amber suppressors derived from a tRNA(Glu)(UUC) gene from D. discoideum. One of the variant tRNA genes codes for a product with a destabilized amino acid acceptor stem. Primary transcripts of this particular su-tRNA(Glu)(CUA) gene are processed only at reduced growth temperatures and consequently function as temperature sensitive suppressors only under these conditions.

Base Sequence↗
PubMed1998

Aminoacylation of hypomodified tRNAGlu in vivo.

The highly specific interaction of each aminoacyl-tRNA synthetase and its substrate tRNAs constitutes an intriguing problem in protein-RNA recognition. All tRNAs have the same overall three-dimensional structure in order to fit interchangeably into the translational apparatus. Thus, the recognition by aminoacyl-tRNA synthetase must be more or less limited to discrimination between bases at specific positions within the tRNA. The hypermodified nucleotide 5-methylaminomethyl-2-thiouridine (mnm5s2U) present at the wobble position of bacterial tRNAs specific for glutamic acid, lysine and possibly glutamine has been shown to be important in the recognition of these tRNAs by their synthetases in vitro. Here, we have determined the aminoacylation level in vivo of tRNAGlu, tRNALys, and tRNA1GIn in Escherichia coli strains containing undermodified derivatives of mnm5s2U34. Lack of the 5-methylaminomethyl group did not reduce charging levels for any of the three tRNAs. Lack of the s2U34 modification caused a 40% reduction in the charging level of tRNAGlu. Charging of tRNALys and tRNA1Gln were less affected. There was no compensating regulation of expression of glutamyl-tRNA synthetase because the relative synthesis rate was the same in the wild-type and mutant strains. These results indicate that the mnm5U34 modification is not an important recognition element in vivo for the glutamyl-tRNA synthetase. In contrast, lack of the s2U34 modification reduced the efficiency of charging by at least 40%. This is the minimal estimate because the turn-over rate of Glu-tRNAGlu was also reduced in the absence of the 2-thio group. Lack of either modification did not affect mischarging or mistranslation.

Base Sequence↗
PubMed1994

Characterization of a cell division gene from Bacillus subtilis that is required for vegetative and sporulation septum formation.

We report the cloning and characterization of a cell division gene, herein designated divIC, from the gram-positive, spore-forming bacterium Bacillus subtilis. This gene was previously identified on the basis of a temperature-sensitive mutation, div-355, that blocks septum formation at restrictive temperatures. We show that the divIC gene is a 125-codon open reading frame that is capable of encoding a protein of 14.7 kDa and that div-355 is a 5-bp duplication near the 3' end of the open reading frame. We also show that divIC is an essential gene by use of an in vitro-constructed null mutation. In confirmation and extension of earlier results, we show that divIC is necessary for both vegetative and sporulation septum formation, and we demonstrate that it is required for the activation of genes expressed under the control of the sporulation transcription factors sigma F and sigma E. The divIC gene is located 1.3 kb upstream of the coding sequence for the sporulation gene spoIIE. Between divIC and spoIIE is a 128-codon open reading frame whose predicted product contains a region of similarity to the RNA-binding domains of polynucleotide phosphorylase and ribosomal protein S1 from Escherichia coli and two putative tRNA genes for methionyl-tRNA and glutamyl-tRNA, the gene order being divIC orf128 tRNA(Met) tRNA(Glu) spoIIE.

Amino Acid Sequence↗