tRNA Tyr Research Articles

Two human tyrosine tRNA genes contain introns

A human λ-phage recombinant which contains at least four tRNA genes, has been isolated. Two DNA fragments were subcloned to give the recombinant plasmids pM6 and pM6128. Nucleotide sequence analysis showed that each plasmid contained a different tyrosine acceptor tRNA (tRNA Tyr) gene. Both tRNA Tyr genes are interrupted by 21-bp introns. These recombinant plasmids have been shown to direct the in vitro synthesis of tRNA-sized products in a HeLa cell transcription system.

Plant tRNA suppressors: In vivo readthrough properties and nucleotide sequence of yellow lupin seeds tRNA Tyr

Transfer RNAs isolated from yellow lupin seeds were screened for tRNA dependent nonsense suppression in Xenopus laevis oocyte system. No opal (UGA) suppressor activity could be detected. However, a tRNA Tyr isoacceptor species stimulates readthrough of the leaky UAG tobacco mosaic virus (TMV)-RNA stop codon. This natural suppressor tRNA was purified to homogeneity and its sequence was determined to be: pCCg ACCUUAm 2GcUCAGDGGm GUAGAGCm 2 2GGAGGACUG ψAm 1GAψCCUUAGm 7Gacp 3UC ACUGGUψCGm 1AAUz.sbnd;CCG GUAGGUCGGACCA OH. The GψA anticodon seqeunce, the presence of the modified nucleoside 3-(3-amino-3-carboxypropyl)uridine (acp 3U) in the extra loo p and the absence of ribothymidine are of special interest.

A fine restriction map of the linear mitochondrial DNA of Tetrahymena pyriformis: genome size, map locations of rRNA and tRNA genes, terminal inversion repeat, and restriction site polymorphism.

A fine restriction map of the linear mitochondrial DNA of Tetrahymena pyriformis strain ST is presented. 1. Based on agarose gel electrophoresis data together with limited nucleotide sequences available on some restriction fragments, we estimate the actual size of this genome to be about 55,000 base pairs. 2. Seven tRNA gene locations have been assigned, which are scattered along the genome length. Six of these locations encode the genes for tRNA(phe), tRNA(his), tRNA(trp), and tRNA(glu), and the duplicate tRNA(tyr) genes which are located at the inverted terminal repeat segments. The tRNA gene(s) encoded in one location has not been identified. We have not yet found the tRNA(leu) and tRNA(met) genes, which were previously shown to be encoded in the genome (Chiu et al. 1974; Suyama 1982). 3. We have mapped the 14S rRNA gene by sequencing the 170 bp segment of EcoRI fragment 8 and by aligning its sequence with E. coli 16S rRNA. From our recent complete sequence data the gene size was found to be about 1,650 bp, which is unexpectedly large for the 14S rRNA which has an estimated size of 1,300 bp. The 14S rRNA is probably a cleavage product of the larger primary transcript of which 200-300 bases of the 5' end are missing. 4. The duplicate copies of the 21S rRNA gene at the terminal duplication inversion segments were analyzed. ClaI fragment 7 (1,500 bp) corresponds in sequence from base position 850 to 2,390 of the 20S rRNA gene of Paramecium mitochondrial DNA (Seilhamer et al. 1984b). The 21S gene is approximately 2,500 bp long. The presence of some restriction site polymorphism is apparent in this segment. 5. Each of the 21S gene copies precedes the tRNA(tyr) gene, but the space flanking one tRNA(tyr) gene differs in size and restriction sites from the space flanking another tRNA(tyr) gene. Thus, this space corresponds to the segment of an imperfect match in the terminal duplication inversion of Goldbach et al. (1978a). 6. Saccharomyces cerevisiae mitochondrial probes including Cob, ATPase VI and IX, and cytochrome oxidase I gene sequences, 21S and 15S rRNAs, and mouse mitochondrial DNA showed no significant hybridization with any restriction fragments of Tetrahymena mitochondrial DNA. The results are in accordance with an extensive sequence divergence previously found in the Tetrahymena mitochondrial genome (Goldbach et al. 1977).

Transfer RNA gene mapping studies on chloroplast DNA from Chlamydomonas reinhardii

Total tRNA of Chlamydomonas reinhardii was fractionated by 2-dimensional gel electrophoresis. Sixteen tRNAs specific for eleven amino acids could be identified by aminoacylation with Escherichia coli tRNA synthetases. Hybridization of these tRNAs with chloroplast restriction fragments allowed for the localization of the genes of tRNA Tyr, tRNA Pro, tRNA Phe (2 genes), tRNA Ile (2 genes) and tRNA His (2 genes) on the chloroplast genome of C. reinhardii. The genes for tRNA Ala (2 genes), tRNA Asn and tRNA Leu were mapped by using individual chloroplast tRNAs from higher plants as probes.

Structural Changes of Yeast tRNATyrCaused by the Binding of Divalent Ions in the Presence of Spermine

The existence of specific sites in tRNA for the binding of divalent cations has been seriously questioned by electrostatic considerations [Leroy & Guéron (1979) Biopolymers, 16, 2429-2446]. However, our earlier studies of the binding of Mg2+ and Mn2+ to yeast tRNA(Tyr) have indicated that spermine creates new binding sites for divalent cations [Weygand-Durasević et al. (1977) Biochim. Biophys, Acta, 479, 332-344; Nöthig-Laslo et al. (1981) Eur. J. Biochem. 117, 263-267]. We have now used yeast tRNA(Tyr), spin labeled at the hypermodified purine (i6A-37) in the anticodon loop, to study the effect of spermine on the binding of manganese ions. The presence of eight spermine molecules per tRNA(Tyr) at high ionic strength (0.2 M NaCl, 0.05 M triethanolamine.HCl) and at low temperature (7 degrees C) enhances the binding of manganese to tRNA(Tyr). This effect could not be explained by electrostatic binding. The initial binding of manganese to tRNA(Tyr) affects the motional properties of the spin label indicating a change of the conformation of the anticodon loop. From the absence of the paramagnetic effect of manganese on the ESR spectra of the spin label one can conclude that the first binding site for manganese is at a distance from i6A-37, influencing the spin label motion through a long-range effect. The enhancement of the binding of manganese to tRNA(Tyr) by spermine is lost upon destruction of its specific macromolecular structure and it does not occur in single stranded or in double-stranded polynucleotides. The observed effect can be explained by the binding of Mn2+ to new sites, created by the binding of spermine, which are specific for the macromolecular structure of tRNA.

The promoter sequence of a yeast tRNA tyr gene

Thirty-one base substitution mutations within the yeast SUP4 tRNA tyr gene were used to probe the effects of different intragenic sequences on promoter activity. The various mutant plasmids were tested quantitatively for their in vitro template activity and for their ability to block competitively the transcription of a reference gene. Five mutations within the coding sequence of SUP4 decreased template activity for pre-tRNA tyr synthesis. The competition assays revealed 11 mutant genes that behaved differently than SUP4-o. Six were weaker competitors and five were stronger. The 12 mutations affecting template activity or competition are clustered in three regions: those encoding the dihydrouracil (D) arm, the extra loop, and the TΨ arm of the tRNA. All of the mutations that reduce competition involve base changes that decrease homology to a eucaryotic tRNA consensus sequence in the highly conserved D and TΨ regions. Three of the five up mutations increased homology to the tRNA consensus sequence.

RNA ligase in bacteria: Formation of a 2′,5′ linkage by an E. coli extract

Ligase activity was detected in extracts of Escherichia coli, Clostridium tartarivorum, Rhodospirillum salexigens, Chromatium gracile, and Chlorobium limicola. Ligase was measured by joining of tRNA halves produced from yeast IVS-containing tRNA precursors by a yeast endonuclease. The structure of tRNA Tyr halves joined by an E. coli extract was examined. The ligated junction is resistant to nuclease P1 and RNAase T2 but sensitive to venom phosphodiesterase and alkaline hydrolysis, consistent with a 2′,5′ linkage. The nuclease-resistant junction dinucleotide comigrates with authentic (2′,5′) A PA marker in thin-layer chromatography. The phosphate in the newly formed phosphodiester bond is derived from the pre-tRNA substrate. The widespread existence of a bacterial ligase raises the possibility of a novel class of RNA processing reactions.

Effects of thyroidectomy on heart and liver rat tRNAs: Study of chromatographic and electrophoretic behaviour

Modifications of tRNAs in various physiological or experimental conditions are well documented. We have compared isoacceptor tRNAs extracted from target organs (heart and liver) from thyroidectomized rats to those of control animals. Nine liver aminoacyl-tRNAs and eight heart aminoacyl-tRNAs from thyroidectomized and control rats were analysed by RPC-5 chromatography. Quantitative differences were demonstrated in the relative proportions of the various liver tRNA isoacceptors for glycine, lysine, methionine, phenylalanine and serine and of the heart isoacceptor tRNAs for glycine, lysine, methionine, phenylalanine and valine. A qualitative variation was noted only for tRNA Tyr from the heart and liver of thyroidectomized rats. Isoacceptor tRNAs were obtained at a high resolution using a two-dimensional polyacrylamide gel electrophoresis. Isoacceptor tRNAs corresponding to 14 amino acids for the liver and 12 amino acids for the heart were identified. Although for most of the tRNAs examined the number of isoacceptors remained unchanged, the number of spots corresponding to tRNA Glu and tRNA His from the liver and tRNA Ala from the heart was different after thyroidectomy. Furthermore the change in electrophoretic behaviour of tRNA Tyr from the liver of thyroidectomized rats suggests a structural modification of one of the isoacceptors in relation to the change in thyroid status. Thus, thyroid hormones appear to induce some modification of the isoacceptor tRNAs in their target organs.

Origin of splice junction phosphate in tRNAs processed by HeLa cell extract

Two cloned tRNA genes that contain intervening sequences, yeast tRNA UCG Ser and Xenopus laevis tRNA Tyr, were transcribed in HeLa cell extract. Precursor tRNAs were formed, and were converted to spliced products by a process of excision-ligation. The novel sequences resulting from ligation of tRNA half-molecules were examined by fingerprinting and nearest neighbor analyses. The results indicate that during tRNA splicing in HeLa cell extract, the 3′-terminal phosphate of the 5′ half-molecule is incorporated into a normal 3′,5′-phosphodiester linkage that forms the splice junction. This ligation pathway in HeLa cell extract is distinct from the one described previously in wheat germ extract, which involves formation of 2′-phosphomonoester, 3′,5′-phosphodiester ▪ linkage with the 3′,5′-bond derived from a 5′-terminal phosphate.

Tumor-associated mutations of rat mitochondrial transfer RNA genes.

Mitochondrial DNA is a sensitive target of chemical carcinogens (Backer and Weinstein (1980) Science 209, 297-299), suggesting that mutations of the mitochondrial genome occur in tumor cells. We examined this point by comparing mitochondrial DNA sequences in four rat tumors with those of normal rat liver. Some novel mutations found in the tRNA genes of tumor mitochondria were as follows: nucleotides deletions in the aminoacyl-acceptor stem of the tRNATyr gene or in the anticodon stem of the tRNATrp gene and insertions in the "YpsiC" loop of the tRNACys gene. These structures are extraordinary compared with those of the tRNA genes of other mammals, indicating that these mutations are each associated with a corresponding tumor.

Functional expression of a yeast ochre suppressor tRNA gene in Escherichia coli

The yeast tRNA Tyr ochre suppressor SUP6 gene and a derivative of this gene in which the 14-bp intervening sequence has been deleted, SUP6Δ, have been examined for functional expression in Escherichia coli. The SUP6Δ, but not the SUP6, gene codes for a functional transfer RNA which has been shown to suppress both ochre and amber nonsense mutations in E. coli. Although the SUP6Δ fragment is contained within a 750-bp restriction fragment, isolated from Saccharomyces cerevisiae, and contains no encoded CCA, the primary transcript, which originates from an E.coli promoter, approx. 1000 bp upstream, is processed to a mature, functional transfer RNA. The pattern of suppression, i.e., suppression of both ochre and amber mutants, is characteristic of E. coli ochre suppressing tRNAs and is in contrast to the pattern observed in yeast, where only ochre mutations are suppressed. The SUP6Δ encoded tRNA, although coding for tRNA Tyr in yeast, is not charged solely with tyrosine in E. coli. The functional expression of this mutant eukaryotic transfer RNA gene in E. coli affords a unique opportunity for studies of expression of a gene coding for a stable RNA, in both a prokaryotic and an eukaryotic host.

Insertion of a repetitive element at the same position in the 5'-flanking regions of two dissimilar yeast tRNA genes.

The regions 5' proximal to many yeast tRNA genes exhibit a high frequency of DNA sequence polymorphisms. DNA sequence analysis of polymorphic variants of SUQ5, a tRNA Ser UCA gene, and SUP2, a tRNA Tyr gene, shows that in each case one sequence variant of the tRNA gene is 346 base pairs longer than the other. The longer variants appear to have arisen from the shorter ones by the insertion of nearly identical copies of a 341-base pair sigma element into a site 16 base pairs upstream from the 5' ends of the tRNA-coding regions. The sequences of the two copies of the sigma element differ at only five positions. The element has a number of properties that are typical of many transposable elements: (i) there is a perfect eight-base-pair inverted repeat at its ends, (ii) these ends are flanked by a five-base-pair direct repeat of a sequence that occurs only once in the target DNA, (iii) there are approximately 20 copies of the element in the yeast genome, and (iv) there is considerable strain-to-strain variation in the sizes of the restriction fragments on which these copies lie. The presence of the sigma element has no gross effect on the phenotype of a SUP2 ochre suppressor. Analysis of the SUQ5 and SUP2 sequences favors the hypothesis that sigma is a transposable element with a novel type of insertion specificity, which is primarily based on the presence of a tRNA-coding region a fixed distance from the insertion site, rather than on the immediate target sequences.

Queuine-containing isoacceptor of tyrosine tRNA in Drosophila melanogaster: Alteration of levels by divalent cations

Dietary cadmium causes the queuine-containing, Q(+), isoacceptors to increase relative to the guanine-containing, Q(−), ones of tRNA Tyr, tRNA His and tRNA Asp of Drosophila melanogaster. Of the other divalent cations examined, Sr 2+, Ni 2+, Cu 2+, Zn 2+ and Hg 2+, only Hg 2+ failed to cause an increase in Q(+)tRNA Tyr. For these results, all pre-adult stages of the organism were spent on media containing the divalent ions. Adult flies that had developed on a normal diet also responsed to divalent ions; Hg 2+ as well as Cd 2+, Sr 2+ and Zn 2+ caused an increase in Q(+)tRNA Tyr in 4 days. Using adult flies, the rate of the response was measured; when placed on a Cd 2+-containing diet, they formed significantly more Q(+)tRNA Tyr within 24 h as compared to adults on a normal diet. Whether the queuine is derived from the diet or from de novo synthesis is yet to be determined. Since the metal ions represent a range of values in the ‘hard-soft’ classification, different sites of reaction are expected, yet for Drosophila a common result is an alteration in the ratio of Q(+) and Q(−) isoacceptors of these tRNAs. The transition to Q(+)tRNA may be an early indication of the metabolic imbalances resulting from the presence of the divalent cation.

Neutron scattering studies of Escherichia coli tyrosyl-tRNA synthetase and of its interaction with tRNA Tyr

Small-angle neutron scattering studies of Escherichia coli tyrosyl-tRNA synthetase indicate that in solution this enzyme is a dimer of M r, 91 (±6) × 10 3 with a radius of gyration R G of 37.8 ± 1.1 Å. The increase in the scattering mass of the enzyme upon binding tRNA Tyr has been followed in 20 m m-imidazole · HCl (pH 7.6), 10 m m-MgCl 2, 0.1 m m-EDTA, 10 m m-2-mercaptoethanol, 150 m m-KCl. A stoichiometry of one bound tRNA per dimeric enzyme molecule was found. The R G of the complex is equal to 41 ± 1 Å. Titration experiments in 74% 2H 2O, close to the matching point of tRNA, show an R G of 38.5 ± 1 Å for the enzyme moiety in the complex. From these values, a minimum distance of 49 Å between the centre of mass of the bound tRNA and that of the enzyme was calculated. In low ionic strength conditions (20 m m-imidazole-HCl (pH 7.6), 10 m m-MgCl 2, 0.1 m m-EDTA, 10 m m-2-mercaptoethanol) and at limiting tRNA concentrations with respect to the enzyme, titrations of the enzyme by tRNA Tyr are characterized by the appearance of aggregates, with a maximum scattered intensity at a stoichiometry of one tRNA per two enzyme molecules. At this point, the measured M r and R G values are compatible with a compact 1:2, tRNA: enzyme complex. This complex forms with a remarkably high stability constant: (enzyme:tRNA:enzyme)/(enzyme:tRNA)(enzyme) of 0.1 to 0.3(× 10 6) m −1 (at 20 °C). Upon addition of more tRNA, the complex dissociates in favour of the 1:1, enzyme:tRNA complex, which has a higher stability constant (1 to 3 (× 10 6) m −1).

Characterization of Glycine max cytoplasmic, chloroplastic and mitochondrial tRNAs and synthetases for phenylalanine, tryptophan and tyrosine

Transfer RNAs and aminoacyl-tRNA synthetases of phenylalanine, tyrosine and tryptophan were localized in the cytoplasm, the chloroplasts and in the mitochondria. The aminoacyl-tRNA synthetases of these three aromatic amino acids fractionated by hydroxylapatite chromatography from both chloroplasts and mitochondria were able to aminoacylate Escherichia coli tRNA suggesting their prokaryotic nature. On the other hand, cytoplasmic enzymes were unable to aminoacylate E. coli tRNA and they were found to be distinct from the organellar aminoacyl-tRNA synthetases in chromatographic behaviour as well as in their kinetic properties. Three isoacceptors for each of tRNA Phe and tRNA Trp were resolved by reversed phase chromatography (RPC-5) and one isoacceptor was localized in each for cytoplasm, chloroplasts and mitochondria. tRNA Tyr was resolved into 5 isoacceptors; 2 were localized in cytoplasm, 2 in chloroplasts and the remaining 1 in mitochondria. Organellar tRNAs in all the three cases could be aminoacylated by E. coli aminoacyl-tRNA synthetases while cytoplasmic tRNAs were not aminoacylatable by E. coli enzymes. Codon recognition studies with tRNA Tyr isoacceptors indicated that all the organellar isoacceptors recognize UAC codon whereas one of the cytoplasmic isoacceptors recognizes UAC codon and the other recognized UAU codon.

A comparative calorimetric study on tRNA unfolding.

The heat effects involved in thermal unfolding of five tRNAs with different primary structures have been determined by direct differential scanning microcalorimetry. The overall molar values of the transition enthalpy (delta Ht) are 1150 kJ/mol for tRNA Lys2 (yeast), 1250 kJ/mol for tRNA Phe (yeast), 1350 kJ/mol for tRNA Val (yeast), 1490 kJ/mol for tRNA Val (E. coli) and 1630 kJ/mol for tRNA Tyr (E. coli). The tRNAs differ in their melting behaviour as can be shown by a comparison of the calorimetric curves. The calorimetrically measured delta Ht values are about 350 kJ/mol higher than the transition enthalpy values for the cloverleaf arrangement, which were estimated using the known parameters for G.C and A.U base pairs.

TRNA synthesis: identification of in vivo precursor tRNAs from parental and mutant yeast strains.

In vivo yeast precursor tRNAs have been identified using a modification of the Northern-hybridization procedure. Two species of pre-tRNA Tyr, 1 species of pre-tRNA Ser2 and 2 species of pre-tRNA Serminor have been found in all yeast strains examined, including parental strains and strains harboring mutations affecting tRNA function. One of the tRNA Tyr strains harboring and one of the pre-tRNA SerUCG are the same size as the unspliced pre-tRNAs which accumulate in the yeast mutant rna1. The in vivo tRNA Tyr precursors detected in these studies also appear similar with the RNA species identified when cloned yeast tRNA Tyr is transcribed and processed by Xenopus oocytes and/or Xenopus extracts. We have also studied the precursor and mature tRNA Tyr species from 22 mutants which contain mutations in the SUP4 tyrosine-inserting suppressor locus. The RNA from 2 mutants mapping at the G52 position showing an aberrantly migrating "mature" tRNA Tyr. Although several of those cloned mutant genes showed transcript products of altered size in in vitro transcription studies (1), we did not detect such altered transcripts in vivo.

RNA processing in microinjected Xenopus oocytes : Sequential addition of base modifications in a spliced transfer RNA

Cloned yeast tRNA Tyr genes are transcribed and processed after micro-injection into Xenopus laevis oocyte nuclei (De Robertis & Olson, 1979; Melton et al., 1980) . The early transcript, about 104 nucleotides long, has a 5′ leader, two or three U residues at the 3′ end and an intervening sequence. The termini are then matured, giving rise to a molecule 92 nucleotides long, which is subsequently spliced to produce a 78 nucleotides long tRNA by a mechanism similar to that found in yeast. Here we analyse the base modifications in these three RNA molecules to test whether the extra RNA segments, and especially the intervening sequence, influence the introduction of base modifications into tRNA. The 104, 92 and 78 RNAs were isolated from gels and analysed by two-dimensional chromatography after complete digestion with nuclease P 1 into 5′-mononucleotides (direct labelling) or ribonuclease T 2 into 3′-mononucleotides (nearest neighbour labelling). By analysing oocytes labelled separately with [α- 32P]ATP, -GTP, -UTP or -CTP it was possible to examine all the base modifications added by the oocyte. The results show that the tRNA Tyr base modifications occur in a sequential order: (1) the early 104 precursor already has five base modifications on it, including all the modifications in the TΨ loop; (2) 11 additional modifications only become apparent in the 92 precursor; and (3) finally, two base modifications, Q base and isopentenyladenosine, are introduced in the anticodon loop only after splicing of the intervening sequence. No modifications were observed in the 5′ leader, 3′ trailer or intervening sequences. Some base modifications added by the oocyte are not normally found in yeast (e.g. Q base), and it seems as if the Xenopus oocyte processes this yeast tRNA as one of its own tRNAs. However, all the modifications normally present in yeast were added by the oocyte, with the exception of a Gm. All base modifications were introduced in a sequential fashion. The splicing pathway found in vitro with yeast extracts (Knapp et al., 1979 ; Peebles et al., 1979) , which has an unusual 3′ phosphate-ended intermediate, was confirmed in vivo in the frog oocyte by analysing the nearest neighbour labelling of the isopentenyladenosine that is located adjacent to the splicing point.

Structure and sequence of the mitochondrial 20S rRNA and tRNA tyr gene of Paramecium primaurelia.

The sequence and structure of the large (20s) mitochondrial (mt) rRNA gene and flanking regions from Paramecium primaurelia have been determined. The gene contains two regions of strong homology with other large mt rRNAs: one 44-base region near the 5' end and a 321-base region near the 3' end. Another region of strong homology to both ends of E. coli 23s RNA exists at loci consistent with these regions. The Paramecium gene appears to be 2204 bases in length and contains slightly more homology to E. coli rRNA than its mammalian or fungal counterparts. The gene, located about 1200 bp from the replicative terminal end of the linear mt DNA, is transcribed in the same polarity as replication. Previous R-looping studies detected no large introns within the gene. Here we describe sequences resembling degenerate rRNAs, one of which could represent a small intron. A tRNA tyr gene was found on the same DNA strand, 127 bp downstream from the large rRNA presumptive 3' end. The tRNA is flanked on both sides by short DNA regions of approximately 90% A + T content.

Mutations of the yeast SUP4 tRNA Tyr Locus: Transcription of the mutant genes in vitro

Twenty-nine different SUP4-o tRNA Tyr genes with second-site mutations were transcribed in X. laevis cell-free RNA polymerase III transcription reactions, and the in vitro transcripts were analyzed by polyacrylamide gel electrophoresis. Nineteen mutant genes yield normal amounts of RNA that co-electrophorese with SUP4-o gene transcripts. RNA synthesized from a mutant gene lacking a single base pair migrated slightly faster in gels, as expected. The still shorter transcripts made from seven other mutant genes suggest that several mutations alter transcription starting or stopping points. Fingerprint analyses of transcripts from the two most extreme cases showed that premature termination occurred at new tracts of T residues resulting from the mutations. Two mutations significantly enhance transcription, and two mutations which alter the invariant C within the TψCG sequence dramatically reduce SUP4-o gene transcription. The regions of the SUP4-o gene that surround these mutations are partially homologous to intragenic sequences in many other eucaryotic tRNA and 5S RNA genes. We hypothesize that these homologous sequences are recognized as promoter regions during RNA polymerase III transcription initiation.