Anticodon loop sequences of transfer RNA Ser CGA and transfer RNA Ser IGA from the posterior silkgland of Bombyx mori L.
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Animals↗
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Exon sequence and structure requirements for tRNA splicing in Saccharomyces cerevisiae.
A survey of exon sequence and structure requirements for splicing was undertaken using labeled pre-tRNA substrates prepared by in vitro transcription of bacterial promoter-yeast tRNA(Tyr) gene fusions. Transcription templates were assembled from oligonucleotide cassettes allowing analysis of 22 derivatives affecting each of the potential secondary and certain tertiary interactions in the pre-tRNA. Effects on both excision of the intervening sequence by yeast endonuclease and joining of exons by ligase were examined. Replacements within the D- and T-stems and anticodon stems revealed that while the primary sequences of these segments were not essential for splicing, formation of base-paired structures was required. Replacements which altered the primary sequence while retaining the secondary structure of the aminoacyl stem allowed efficient excision by endonuclease but reduced joining by ligase. Potentially, the effects of changes within these stems may be indirect through effects on adjacent or overall structure. The presence of either structured or unstructured 5' leader and/or 3' trailer sequences had no effect on either splicing step. Alterations in the conserved Levitt tertiary pair (G15/C48), previously implicated in splicing of pre-tRNA(Phe), did not alter splicing of pre-tRNA(Tyr). A precursor in which the small (type I) extra arm in pre-tRNA(Tyr) was replaced with the large (type II) extra arm sequence from tRNA(Ser) was efficiently spliced. These and previous results suggest that only limited features of exon sequence or structure are recognized by the splicing enzymes.
Base Sequence↗
Recognition nucleotides of Escherichia coli tRNA(Leu) and its elements facilitating discrimination from tRNASer and tRNA(Tyr).
In order to study how Escherichia coli leucyl-tRNA synthetase recognizes tRNA(Leu) and discriminates it from the other two class II tRNAs, tRNA(Ser) and tRNA(Tyr), various mutations were introduced into class II tRNA transcripts. The discriminator base A73, but not the anticodon sequence, was found to serve as a critical recognition element of tRNA(Leu). A base substitution at the invariant nucleotide A14, but not at any of the other nucleotides characteristic of the E. coli tRNA(Leu) isoacceptors among the three class II tRNAs, caused significantly damaged aminoacylation with leucine. A two base-pair deletion in the long variable arm also resulted in no significant decrease of activity. Transplanting the three tertiary elements characteristic of E. coli tRNA(Leu) (i.e. the location of the G18G19 sequence in the D-loop, the A15 U48 base-pair and the stem pairing pattern of the long variable arm) besides the discriminator base change introduced the leucine charging activity in terms of Vmax/Km, up to 0.1 of that for the normal sequence of tRNA(Leu) into both tRNA(Ser) and tRNA(Tyr). These results indicate that A73 and A14 (or its vicinity) are involved in recognition by leucyl-tRNA synthetase, and that several tertiary elements play a significant role in the discrimination of tRNA(Leu) from the other two class II tRNAs.
Base Sequence↗
Identity determinants of E. coli tRNAs.
Identity determinants of E. coli tRNA(Val) and three class II tRNAs, tRNA(Ser), tRNA(Tyr) and tRNA(Leu), are studied by using various variants of tRNA transcripts. Anticodon, discriminator base and acceptor stem are involved in the identity elements for tRNA(Val). Discrimination among class II tRNAs are considered to be dependent on the bases at positions 2, 71 and 73 as well as their different tertiary structures including the long variable arm.
Codon↗
An evolutionary 'intermediate state' of mitochondrial translation systems found in Trichinella species of parasitic nematodes: co-evolution of tRNA and EF-Tu.
EF-Tu delivers aminoacyl-tRNAs to ribosomes in the translation system. However, unusual truncations found in some animal mitochondrial tRNAs seem to prevent recognition by a canonical EF-Tu. We showed previously that the chromadorean nematode has two distinct EF-Tus, one of which (EF-Tu1) binds only to T-armless aminoacyl-tRNAs and the other (EF-Tu2) binds to D-armless Ser-tRNAs. Neither of the EF-Tus can bind to canonical cloverleaf tRNAs. In this study, by analyzing the translation system of enoplean nematode Trichinella species, we address how EF-Tus and tRNAs have evolved from the canonical structures toward those of the chromadorean translation system. Trichinella mitochondria possess three types of tRNAs: cloverleaf tRNAs, which do not exist in chromadorean nematode mitochondria; T-armless tRNAs; and D-armless tRNAs. We found two mitochondrial EF-Tu species, EF-Tu1 and EF-Tu2, in Trichinella britovi. T.britovi EF-Tu2 could bind to only D-armless Ser-tRNA, as Caenorhabditis elegans EF-Tu2 does. In contrast to the case of C.elegans EF-Tu1, however, T.britovi EF-Tu1 bound to all three types of tRNA present in Trichinella mitochondria. These results suggest that Trichinella mitochondrial translation system, and particularly the tRNA-binding specificity of EF-Tu1, could be an intermediate state between the canonical system and the chromadorean nematode mitochondrial system.
Amino Acid Sequence↗
Genes for tRNA(Gly), tRNA(His), tRNA(Lys), tRNA(Phe), tRNA(Ser) and tRNA(Tyr) are encoded in Oenothera mitochondrial DNA.
The genes coding for tRNA(Gly), tRNA(His), tRNA(Lys), tRNA(Phe), tRNA(Ser) and tRNA(Tyr) have been identified in Oenothera mitochondrial DNA. Sequence analysis of these genes and their surrounding sequences are presented and compared with other known tRNA genes from plant mitochondria. All six deduced tRNA sequences can be folded into the classical cloverleaf structure model. Only the tRNA(His) gene shows high homology with the corresponding chloroplast gene and thus appears to be derived from a transfer event of chloroplast sequences into the mitochondrial genome. The sequences surrounding this gene, however, show little similarity with the chloroplast genome. The other five deduced tRNAs display a much lower similarity with their chloroplast counterparts and thus appear to be genuine mitochondrial tRNAs. These tRNAs are highly conserved between monocots and dicots with maximally three nucleotides differing between the Oenothera sequences and their wheat homologues. A purine-rich sequence is found upstream of each tRNA gene in Oenothera, similar to wheat mitochondrial tRNA genes, that could be involved in transcription signalling.
Base Sequence↗
Discrimination among E. coli tRNAs with a long variable arm.
In E. coli, tRNA(Ser), tRNA(Leu) and tRNA(Tyr) have a long variable arm composed of more than ten nucleotides (class II tRNAs). In order to study how leucyl- and seryl-tRNA synthetase discriminate their cognate tRNA isoacceptors from the other class II tRNAs, kinetic parameters of various mutated class II tRNA transcripts with leucyl- and seryl-tRNA synthetase were determined. Leucyl-tRNA synthetase recognizes A73 and A14 or its vicinity. Seryl-tRNA synthetase recognizes the long variable arm base-nonspecifically. C2-G71 in the acceptor stem functions as a negative identity element against seryl-tRNA synthetase. Difference in the tertiary structure among class II tRNA molecules plays a crucial role in discrimination by these two synthetases.
Acylation↗
Novel autoantibodies directed against the common tertiary configuration of transfer RNA in a patient with interstitial lung disease.
OBJECTIVE: To identify and characterize a novel autoantibody, anti-WS, that binds total transfer RNA (tRNA). METHODS: Serum from patient WS, who had polyarthritis, Sjögren's syndrome, Raynaud's phenomenon, and interstitial pulmonary fibrosis, was used in this study. Characteristics of anti-WS and antibody-reactive determinants of tRNA were investigated by 32P immunoprecipitation using HeLa cell RNA and deletion mutants of tRNA transcribed in vitro. RESULTS: WS serum produced nucleolar and cytoplasmic staining on indirect immunofluorescence. 32P immunoprecipitation assays demonstrated that this serum immunoprecipitated total tRNAs and 5.8S and 5S ribosomal RNAs from 32P-labeled HeLa cell extract. When deproteinized RNA was used as antigen source, total tRNAs were still precipitated by WS serum. An immunoprecipitation study, using various deletion mutants of Escherichia coli tRNA, demonstrated that both D and T psi C loops were needed for antibody binding. Substitution of nucleotide 18G with 18A of E coli tRNA(Trp), which is essential in the formation of the tertiary "L" shape of tRNA, inhibited binding by anti-WS antibodies. CONCLUSION: Anti-WS antibodies are novel autoantibodies directed against tRNAs. The antibody binding site is the common L-shaped tertiary structure conformed by the D loop and T psi C loop of tRNA, suggesting that the antibodies are induced by a conserved sequence among all species. Furthermore, these antibodies could be a marker for a newly recognized subset of connective tissue disease.
Autoantibodies↗
Molecular phylogenetics of emydine turtles: taxonomic revision and the evolution of shell kinesis.
The 10 extant species of emydine turtles represent an array of morphological and ecological forms recognizable and popular among scientists and hobbyists. Nevertheless, the phylogenetic affinities of most emydines remain contentious. Here, we examine the evolutionary relationships of emydine turtles using 2092 bp of DNA encoding the mitochondrial genes cyt b, ND4, and adjacent tRNAs. These data contain 339 parsimony informative characters that we use to erect hypotheses of relationships for the Emydinae. Both maximum parsimony and maximum likelihood methods yield a monophyletic Emydinae in which all but three nodes are well resolved. Emys orbicularis, Emydoidea blandingii, and Clemmys marmorata form a monophyletic clade, as do the species of Terrapene. Clemmys muhlenbergii and Clemmys insculpta form a third monophyletic group that may be sister to all other emydines. Clemmys guttata is problematic and probably related to Terrapene. Based on this phylogeny, and previous molecular work on the group, we suggest the following taxonomic revisions: (1) Clemmys should be restricted to a single species, C. guttata. (2) Calemys should be resurrected for C. muhlenbergii and C. insculpta. (3) Emys should be expanded to include three species: E. orbicularis, E. blandingii, and E. marmorata. Furthermore, our analyses show that neither kinetic-shelled nor akinetic-shelled emydines form monophyletic groups. Therefore, shell kinesis was either independently gained in Emys and Terrapene or secondarily lost in E. marmorata and C. guttata. Parsimony, paleontological evidence, and the multiple origins of shell kinesis in related turtle lineages (especially geoemydines) support the independent origin of plastral kinesis.
Animals↗
Rapid mapping of markers applying vectorette technology to YAC fragmentation allows easy assembly of a high-density STS bacterial clone contig spanning the markers D6S1260-D6S1918.
We have generated a detailed physical map of the 6p21.3/p22.1 boundary, using a combination of yeast artificial chromosome (YAC) fragmentation and high-resolution sequence tagged site (STS) content mapping. YACs from the CEPH, St. Louis, and ICRF libraries have been used to construct a 4.5-Mb contig spanning the markers D6S306 to D6S1571. YAC insert sizes were determined by pulsed field gel electrophoresis (PFGE). Chimerism of YACs was determined by fluorescent in situ hybridization (FISH), and their integrity was determined by fingerprinting with Alu-PCR. We have identified 10 new CA repeat loci in this region as well as over 50 novel STSs, several tRNA genes, a new histone H2B gene and the phospholipase D gene. Using these new markers, we have rapidly generated a bacterial clone contig of over 250 kb, spanning the markers D6S1260 to D6S1918 (WI-3111) with STSs spaced on average every 6 kb.
Animals↗
Dual hydrolytic role for Pb(II) ions.
RNA phosphodiester bonds can be cleaved by metal ions, of which Pb2+ is one of the most effective. It can cleave both generally and site-specifically, depending on the substrate and the conditions. In addition, metal ions are also known to cleave ester bonds between amino acid and the 3'-end of transfer RNA. Here we report that in aminoacylated transfer RNA, Pb2+ ions cleave internucleotide bonds in the 3'-end of tRNA and also cleaves the bond between tRNA and its amino-acid, attached at the 3'-end via an ester bond to the terminal ribose in aminoacyl tRNA. The two reactions proceed at different rates. The rate of deacylation is significantly faster than the rate of cleavage of phosphodiester bonds, with a pH-optimum of 7. This dual hydrolytic role is not seen for other metal ions examined, namely Zn(II), Cd(II) and Mn(II). The rate of the two kinds of hydrolyses by Pb2+ ions is compared with that of other metal-ions. The mechanism of cleavage is investigated further by modification of the 3'-end of tRNA.
Acylation↗
Achieving error-free translation; the mechanism of proofreading of threonyl-tRNA synthetase at atomic resolution.
The fidelity of aminoacylation of tRNA(Thr) by the threonyl-tRNA synthetase (ThrRS) requires the discrimination of the cognate substrate threonine from the noncognate serine. Misacylation by serine is corrected in a proofreading or editing step. An editing site has been located 39 A away from the aminoacylation site. We report the crystal structures of this editing domain in its apo form and in complex with the serine product, and with two nonhydrolyzable analogs of potential substrates: the terminal tRNA adenosine charged with serine, and seryl adenylate. The structures show how serine is recognized, and threonine rejected, and provide the structural basis for the editing mechanism, a water-mediated hydrolysis of the mischarged tRNA. When the adenylate analog binds in the editing site, a phosphate oxygen takes the place of one of the catalytic water molecules, thereby blocking the reaction. This rules out a correction mechanism that would occur before the binding of the amino acid on the tRNA.
Amino Acid Sequence↗
Accumulation of nuclear-encoded tRNA(Thr) (AGU) in mitochondria of the liverwort Marchantia polymorpha.
The mitochondrial genome of the liverwort Marchantia polymorpha does not encode the full complement of tRNAs for the threonine and isoleucine codon boxes. To find the missing tRNA genes specifically for tRNA(Thr) in mitochondria, we have searched the genomic library and identified two clones (pTT1 and pTT2), encoding the identical tRNA(Thr) (AGU) gene copy with different 5'- and 3'-flanking sequences. By northern analysis, we demonstrate considerable accumulation of the nuclear encoded tRNA(Thr) and moderate expression of native tRNA(Thr) (GGU) in mitochondria. Nonetheless, the imported and native tRNA(Thr) species together are not sufficient to translate all four threonine codons used in liverwort mitochondria, implicating mitochondrial import of at least one additional threonine isoacceptor tRNA.
Anticodon↗
Factor for inversion stimulation-dependent growth rate regulation of serine and threonine tRNA species.
We have previously shown that the accumulation of 20 tRNA species in Escherichia coli is individually regulated as a function of cellular growth rate. We have also reported that the growth rate regulation of some but not all tRNA species is dependent on the activity of the factor for inversion stimulation (FIS). In present work, we studied the growth rate regulation of the serine- and threonine-accepting tRNA families. We show that the levels of tRNA(3Thr), tRNA(3Ser), tRNA(2Thr), tRNA(3Thr), and tRNA(4Thr) are reduced in fis cells as the growth rate increases. The accumulation of these tRNA species is reduced 2-5-fold at the fastest bacterial growth rate. The strongest effect is observed for the two minor tRNA species; tRNA(2Ser) and tRNA(2Thr). In contrast, we find that the accumulation of tRNA(1Ser), tRNA(5Ser), and tRNA(1Thr) is similar in wild type and fis bacteria. The data presented provide further evidence for the suggestion that FIS is a stimulating factor that is involved, directly or indirectly, in the high expression level of some tRNA genes at fast bacterial growth rates.
Base Sequence↗
Processing and editing of overlapping tRNAs in human mitochondria.
Overlapping tRNA genes in mitochondria of many metazoans introduce a problem for the processing of such polycistronic primary transcripts. Using runoff transcripts and an S100 extract from HeLa cell mitochondria, the processing of the human mitochondrial tRNATyr/tRNACys precursor (carrying an overlap of one base) was investigated: tRNACys is released in its complete form carrying the overlapping residue at the first position, whereas tRNATyr lacks that nucleotide at the discriminator position. Partial deletion of tRNACys or complete replacement by a non-tRNA-like sequence does not alter the processing reaction and indicates that the upstream tRNATyr alone is recognized by a 3'-endonuclease activity. The truncated 3'-end of this tRNATyr is then completed in an editing reaction that incorporates the missing residue. The processing of this tRNA overlap seems to be species-specific, because an overlapping tRNA precursor (tRNASer(AGY)/tRNALeu(CUN)) from opossum mitochondria is not recognized by the human extract. Because processing activities for overlapping and nonoverlapping tRNA precursors could not be separated, it seems that one general activity is responsible for the 3'-end processing of mitochondrial tRNAs and that this activity coevolved with the particular overlap between tRNATyr and tRNACys in human mitochondria, being unable to recognize overlaps between other tRNAs.
Base Sequence↗
Suppression of single and double nonsense mutations introduced into the diphtheria toxin A-chain gene: a potential binary system for toxin gene therapy.
We have previously shown that ablation of specific cells can be achieved through the transcriptionally regulated expression of the diphtheria toxin A-chain (DT-A) gene in both cell culture and transgenic mice. Such targeted toxin gene expression provides a novel approach to cancer and acquired immunodeficiency syndrome (AIDS) therapy. The use of mutants of DT-A with attenuated toxicity may allow targeting of cells for which only moderately selective gene regulatory elements are available. Alternatively, conditional mutants might be used to target cells in which conditions can be established for suppression of the mutation. We have investigated the effects of mutating selected serine codons to amber (TAG) nonsense codons in the DT-A coding sequence. In transient transfection of HeLa cells, DT-A activity was markedly reduced by the introduction of a single amber codon and was virtually eliminated by two amber mutations. Cotransfection of a serine inserting suppressor tRNA expression plasmid substantially restored DT-A expression from both single and double amber mutants. Expression of the same suppressor tRNA also suppressed a previously described amber mutation at the tyrosine codon 28 in DT-A. Thus, nonsense suppression can be used to control the expression of DT-A in mammalian cells, potentially allowing binary control over the targeting of tissues for selective ablation.
Amino Acid Sequence↗
Effect of B. subtilis TRNA(Trp) on readthrough rate at an opal UGA codon.
Bacillus subtilis has been thought to have a high readthrough rate at the UGA stop codon because no opal suppressor tRNA has been isolated so far [Lovett et al. (1991) J. Bacteriol. 173, 1810-1812]. To examine whether a tRNATrp which we have characterized [Matsugi et al. (1992) Nucleic Acids Res. 20, 3514] has the ability to read the UGA codon, in vitro translation was performed with a synthetic mRNA containing a test codon, UGA, UAG, UAA, or UGG, in a reading frame. Addition of Trp-tRNATrp to the system significantly increased the readthrough rate only in the case of UGA. This suggests that this tRNATrp has a dual recognition pattern in B. subtilis, i.e., for the canonical tryptophan codon and for readthrough at the UGA stop codon.
Anticodon↗
Sequence-specific binding of transfer RNA by glyceraldehyde-3-phosphate dehydrogenase.
A transfer RNA (tRNA) binding protein present in HeLa cell nuclear extracts was purified and identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Studies with mutant tRNAs indicated that GAPDH recognizes both sequence and structural features in the RNA. GAPDH discriminated between wild-type tRNA and two tRNA mutants that are defective in nuclear export, which suggests that the protein may participate in RNA export. The cofactor nicotinamide adenine dinucleotide disrupted complex formation between tRNA and GAPDH and thus may share a common binding site with the RNA. Indirect immunofluorescence experiments showed that GAPDH is present in the nucleus as well as in the cytoplasm.
Amino Acid Sequence↗