Transfer RNA determinants for translational editing by Escherichia coli valyl-tRNA synthetase

Valyl-tRNA synthetase (ValRS) has difficulty differentiating valine from structurally similar non-cognate amino acids, most prominently threonine. To minimize errors in aminoacylation and translation the enzyme catalyzes a proofreading (editing) reaction that is dependent on the presence of cognate tRNA^Val. Editing occurs at a site functionally distinct from the aminoacylation site of ValRS and previous results have shown that the 3′-terminus of tRNA^Val is recognized differently at the two sites. Here, we extend these studies by comparing the contribution of aminoacylation identity determinants to productive recognition of tRNA^Val at the aminoacylation and editing sites, and by probing tRNA^Val for editing determinants that are distinct from those required for aminoacylation. Mutational analysis of Escherichia coli tRNA^Val and identity switch experiments with non-cognate tRNAs reveal a direct relationship between the ability of a tRNA to be aminoacylated and its ability to stimulate the editing activity of ValRS. This suggests that at least a majority of editing by the enzyme entails prior charging of tRNA and that misacylated tRNA is a transient intermediate in the editing reaction.     

RNA-DNA hybridization analyses of tRNA-Val-3b in Drosophila melanogaster

Summary Transfer RNA was extracted from 50–300 mg of adult flies and specifically labeled in vitro. The level of individual isoacceptors was quantitated by efficient annealing to Drosphila tRNA genes carried on recombinant DNA plasmids immobilized on nitrocellulose filters. The level of tRNA _3b ^Val in the tRNA isolated from flies deficient in the major tRNA _3b ^Val loci has been examined. The results show that deletion of the major tRNA _3b ^Val loci resulted in a reduction of approximately 50% in the level of tRNA _3b ^Val but did not produce the Minute phenotype; furthermore the effects of deficiencies at two loci were approximately additive.     

Genes coding for valine transfer ribonucleic acid-3b in Drosophila melanogaster.

The genes for tRNA_3b^val were localized to 84D and 92B on the polytene chromosomes of Drosophila melanogaster with a possible minor site at 90B-C by hybridization in situ and autoradiography with ¹²⁵I-labeled tRNA_3b^val. Flies carrying a duplication of the 84D region had increased amounts (30%) of tRNA_3b^val in proportion to the increased number of genes. While a proportional decrease in the amount of tRNA^val_3b in flies bearing a deletion of the same region was found, the total acceptance of valine remained at the level found in the wild type.     

Genes for tRNALys5 from Drosophila melanogaster.

The sequences of two cloned genes from Drosophila which hybridize with tRNALys5 are reported. One gene, in plasmid pDt39, has a sequence which corresponds to the sequence of tRNA. The other gene, in pDt59R, differs in three nucleotides pairs. Both plasmids are transcribed in vitro with extracts of Drosophila Kc cells to give full-sized tRNA precursors with four additional nucleotides at the 5'-end as well as truncated molecules containing 35 nucleotides. This premature termination occurs in a block of four T residues within the mature coding region. Sequences flanking the tRNA genes show little in common except for the blocks of five or more T-residues beyond the 3'-end of the gene. pDt39 hybridizes to 84AB on the polytene chromosomes of Drosophila and pDt59R hybridizes to 29A.     

The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC)

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo⁵UAC) contains a unique modification, N⁶-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA₁^Val. Inactivation of yfiC gene abolishes m⁶A formation in tRNA₁^Val, while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA₁^Val can be methylated by recombinant YfiC protein in vitro. Although the methylation of m⁶A in tRNA₁^Val by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.     

Evaluation of a novel 16S rRNA/tRNA mitochondrial marker for the identification and phylogenetic analysis of shrimp species belonging to the superfamily Penaeoidea

In this study, we infer the phylogenetic relationships within commercial shrimp using sequence data from a novel mitochondrial marker consisting of an approximately 530-bp region of the 16S ribosomal RNA (rRNA)/transfer RNA (tRNA)^Val genes compared with two other mitochondrial genes: 16S rRNA and cytochrome c oxidase I (COI). All three mitochondrial markers were considerably AT rich, exhibiting values up to 78.2% for the species Penaeus monodon in the 16S rRNA/tRNA^Val genes, notably higher than the average among other Malacostracan mitochondrial genomes. Unlike the 16S rRNA and COI genes, the 16S rRNA/tRNA^Val marker evidenced that Parapenaeus is more closely related to Metapenaeus than to Solenocera, a result that seems to be more in agreement with the taxonomic status of these genera. To our knowledge, our study using the 16S rRNA/tRNA^Val gene as a marker for phylogenetic analysis offers the first genetic evidence to confirm that Pleoticus muelleri and Solenocera agassizi constitute a separate group and that they are more related to each other than to genera belonging to the family Penaeidae. The 16S rRNA/tRNA^Val region was also found to contain more variable sites (56%) than the other two regions studied (33.4% for the 16S rRNA region and 42.7% for the COI region). The presence of more variable sites in the 16S rRNA/tRNA^Val marker allowed the interspecific differentiation of all 19 species examined. This is especially useful at the commercial level for the identification of a large number of shrimp species, particularly when the lack of morphological characteristics prevents their differentiation.     

Interactions of yeast valyl-tRNA synthetase with RNAs and conformational changes of the enzyme.

Different conformations have been identified for the enzyme valyl-tRNA synthetase from yeast inside its complex with one tRNA molecule by neutron scattering. One form is identical to that of the free enzyme in solution; the other form is more contracted, having a radius of gyration which is smaller by 10% and a specific volume which is smaller by 1%. The contracted conformation has been found for the complexes with tRNA^Val and tRNA^Asp in phosphate buffer (pH 6.3) provided the ionic strength is lower than about 150 mm. In higher ionic strength (up to about 500 mm) the enzyme still forms a complex with tRNA^Val but its conformation remains that of the free protein in solution. In the complex with tRNA₃^Leu, the enzyme conformation is that of the free state even at the lowest ionic strength examined (that of the phosphate buffer, 60 mm). The free enzyme is an elongated molecule of radius of gyration 40 Å (a compact protein of the same molecular weight would have a radius of gyration of 30 Å).The positioning within the complex of tRNA^Val, on the one hand, and tRNA₃^Leu, on the other, is very different. The first tRNA is intimately associated with the enzyme, lying predominantly closer to the centre of mass of the complex than the protein. In the complex with tRNA₃^Leu, the tRNA lies further away from the centre of mass of the complex than the protein.Small concentrations of tRNA^Val, tRNA^Asp, tRNA₃^Leu or Escherichia coli 5 S ribosomal RNA cause the enzyme to aggregate into dimers, trimers and higher aggregates provided the ionic strength of the buffer is below 150 mm. In higher ionic strength or for [RNA]: [enzyme] > 1 the aggregates are dissociated to yield the one-to-one RNA-enzyme complex.     

The yeast cloning vector YEp13 contains a tRNA3Leu gene that can mutate to an amber suppressor

We have shown that the yeast-Escherichia coli shuttle vector YEp 13 contains, as part of its yeast chromosomal segment, a tRNA₃^Leu gene. We have also isolated and characterized a variant of YEp13, namely YEp13-a, which is capable of suppressing a variety of yeast amber-suppressible alleles in vivo. YEp13-a differs from YEp13 by a single point mutation, which changes the three-nucleotide, plus-strand sequence corresponding to the tRNA₃^Leu anticodon from the normal C-A-A to C-T-A. This nucleotide change creates a site for the restriction enzyme XbaI in the suppressor tRNA₃^Leu gene. We have taken advantage of the correlation between the suppressor mutation and the XbaI site formation, to show that the tRNA₃^Leu gene on YEp13 corresponds to the genetically characterized yeast chromosomal amber suppressor SUP53. We have also shown that SUP53 is located just centromere-distal to LEU2 on chromosome III. Finally, comparison of the DNA sequence of SUP53 and its flanking regions with the sequences of other cloned yeast tRNA₃^Leu genes has revealed considerable sequence homology in the immediate 5′-flanking regions of these genes.     

Activation of several tRNAs of Escherichia coli by the phenoxyacetyl derivative of N-hydroxysuccinimide

Escherichia coli 15T^? treated with chloramphenicol produces tRNA^phe which is deficient in minor nucleosides. Undermodified tRNA^phe chromatographs as two new peaks from a benzoylated diethylaminoethyl-cellulose column. Chloramphenicol tRNA^phe was purified by phenoxyacetylation of phenylalanyl-tRNA and subsequent chromatography on benzoylated diethylaminoethyl-cellulose. Purified tRNA^phe had an altered Chromatographie profile as a result of the purification procedure. Phenoxyacetylation of an unpurified tRNA preparation, which was either charged with phenylalanine or kept discharged, resulted in a permanent alteration of tRNA^phe which was similar to the alteration of the purified tRNA^phe. The altered tRNAs eluted with higher salt or ethanol concentrations from benzoylated diethylaminoethyl-cellulose. The alteration was also shown for tRNA^phe of phenoxyacetylated tRNA from late log phase E. coli 15T?. tRNA^glu and tRNA^Leu were not changed, but both tRNA^Arg and tRNA^Ile were altered. tRNA₂^Val and tRNA^Met shifted in the elution profile; tRNA₁^Val and tRNA_f^Met were not affected.Comparison of the primary structures of the alterable and nonalterable tRNA's revealed that all alterable tRNA's have the undefined nucleoside X in the extra loop. Phenoxyacetylation of nucleoside X probably was the cause of the altered profiles.tRNA^phe from E. coli 15T^? treated with chloramphenicol was less reactive towards phenoxyacetylation than normal tRNA, possibly because of a different conformation of the modification-deficient molecule relative to the normal tRNA^phe. tRNA^phe from E. coli 15T^?, starved for cysteine and methionine and treated with chloram-phenicol, is more deficient in minor nucleosides and showed even less reactivity.Acceptor capacities of the altered tRNA species were not changed significantly; only the acceptor capacity for tRNA^Ile decreased approximately 25%. The recognition site for the aminoacyl-tRNA synthetases probably is not affected.     

The anticodon-anticodon complex

Gel electrophoresis has been used to measure the binding between two tRNAs with complementary anticodons, tRNA^Val (Escherichia coli) (anticodon X,A,C) and tRNA^Tyr (E. coli) (anticodon Q,U,A). The association constant K at 0 °C was found to be 4 × 10⁵ m^?1 which is about three orders of magnitude greater than the association constant for tRNA^Tyr (E. coli) binding its trinucleotide codon UAC. The temperature dependence of K suggests that this results from the rigidity of the anticodon loop. tRNA^Tyr (E. coli) binds an order of magnitude more weakly to tRNA^Val (yeast) than to tRNA^Val (E. coli), presumably because it contains the wobble base pair A · I. The relationship between the anticodon-anticodon complex and codon recognition is discussed.     

Analysis of purified tRNA species by polyacrylamide gel electrophoresis

Six purified amino acid acceptor tRNA species were examined by polyacrylamide gel electrophoresis. Small differences in migration were observed under conditions that preserve the conformation of tRNA. When tRNA was heated in the presence of either 10 mM acetate or EDTA at 60° a change in migration was observed for tRNA^Glu. No difference in migration was seen between Val-tRNA^Val and tRNA^Val. When tRNA was denatured by heating in 4M urea and applied to a gel containing the same amount of urea, all tRNA species migrated approximately the same distance with the exception of tRNA^{Leu V}, which showed an appreciable slower migration. From the difference in migration of tRNA^{Leu V} as compared to tRNA^Val and 5 S RNA, the difference in chain length between tRNA^Val and tRNA^{Leu V} was estimated to be approximately 9 nucleotides.     

Photochemistry of 4-thiouridine in Escherichia coli transfer RNA1Val

Irradiation of pure transfer RNA₁^Val with monochromatic light (334 nm) produces characteristic changes in the spectral properties of 4-thiouridine, the only base which strongly absorbs light at this wavelength. Variations in absorption and fluorescence of 4-thiouridine during irradiation are interpreted in terms of a specific, quantitative photoreaction which proceeds with a yield of about 5 × 10⁻³E/mole. The photoreaction occurs under conditions where tRNA₁^Val is biologically active but not under conditions that destroy the tertiary structure of the 4-thiouridine region.     

Localization of four genes for three tRNALeu's on the physical map of the soybean (Glycine max L.) chloroplast genome

Three tRNAs^Leu from soybean chloroplasts were isolated and hybridized to restriction fragments of soybean chloroplast DNA. Based on the hybridization pattern, the locations of four genes coding for tRNA₁^Ley, tRNA₂^Leu (two genes tRNA_2a^Ley and tRNA_2b^Leu, are present in the inverted repeat region) and tRNA₃^Leu were determined on the physical map of the soybean chloroplast genome.     

Cloning, Sequencing and Analysis of the 16S-23S rDNA Intergenic Spacers (IGSs) of Two Strains of Vibrio vulnificus

According to the conserved sequences flanking the 3′ end of the 16S and the 5′ end of the 23S rDNAs, PCR primers were designed, and the 16S-23S rDNA intergenic spacers (IGSs) of two strains of Vibrio vulnificus were amplified by PCR and cloned into pGEM-T vector. Different clones were selected to be sequenced and the sequences were analyzed with BLAST and the software DNAstar. Analyses of the IGS sequences suggested that the strain ZSU006 contains five types of polymorphic 16S-23S rDNA intergenic spacers, namely, IGS^GLAV, IGS^GLV, IGS^lA, IGS^G and IGS^A; while the strain CG021 has the same types of IGSs except lacking IGS^A. Among these five IGS types, IGS^GLAV is the biggest type, including the gene cluster of tRNA^Glu - tRNA^Lys - tRNA^Ala - tRNA^Val; IGS^GLV includes that of tRNA^Glu-tRNA^Lys-tRNA^Val; IGS^AG, tRNA^Ala-tRNA^Glu; IGS^IA, tRNA^Ile-tRNA^Ala; IGS^G, tRNA^Glu and IGS^A, tRNA^Ala. Intraspecies multiple alignment of all the IGS sequences of these two strains with those of V. vulnificus ATCC27562 available at GenBank revealed several highly conserved sequence blocks in the non-coding regions flanking the tRNA genes within all of strains, most notably the first 40 and last 200 nucleotides, which can be targeted to design species-specific PCR primers or detection probes. The structural variations of the 16S-23S rDNA intergenic spacers lay a foundation for developing diagnostic methods for V. vulnificus.     

Clustering of transfer RNA genes of Xenopus laevis

The arrangement of the reiterated DNA sequences complementary to transfer RNA has been studied in Xenopus laevis. Prehybridization of denatured DNA with an excess of unfractionated tRNA results in a small but well-defined increase in the buoyant density of fragments which contain sequences homologous to tRNA. The density increase is smaller than that found for 5 S DNA, but is the same or nearly so for all tRNA coding sequences examined. These results indicate that the majority of tRNA genes are clustered together with spacer DNA, the average size of which is estimated to be approximately 0.5 × 10⁶ daltons (native) DNA.In high molecular weight native DNA preparations, the sequences homologous to unfractionated tRNA, tRNA^Val, tRNA₁^Met and tRNA₂^Met band in CsCl at 1.707, 1.702, 1.708 and 1.711 g cm^?3, respectively. The mean buoyant densities are constant at all molecular weights examined but they do not correspond to the base compositions of the complementary tRNA species. These results indicate that isocoding genes are linked to spacer DNA in separate and extensive gene clusters, and that the different clusters contain different spacer DNA sequences. These clusters form well-defined cryptic DNA satellites which are potentially separable from each other as well as from other chromosomal DNA.     

Nucleotide sequences of yeast genes for tRNA(2), tRNA(2) and tRNA(1): homology blocks occur in the vicinity of different tRNA genes

Three members of a collection of pBR322-yeast DNA recombinant plasmids containing yeast tRNA genes have been analyzed and sequenced. Each plasmid carries a single tRNA gene: pY44, tRNA^Ser₂; pY41, tRNA^Arg₂; pY7, tRNA^Val₁. All three genes are intronless and terminate in a cluster of Ts in the non-coding strand. The sequence information here and previously determined sequences allow an extensive comparison of the regions flanking several yeast tRNA genes. This analysis has revealed novel features in tRNA gene arrangement. Blocks of homology in the flanking regions were found between the tRNA genes of an isoacceptor family but, more interestingly, also between genes coding for tRNAs of different amino-acid specificities. Particularly, three examples are discussed in which sequence elements in the neighborhood of different tRNA genes have been conserved to a high degree and over long distances.     

Half molecules of serine-specific transfer ribonucleic acids from yeast

The preparation and analysis of half molecules from tRNA^Ser are described. Two pG-halves were isolated which differed only in the presence or absence of an acetyl group on the cytidylic acid residue at position 12. The CCA-half derived from tRNA₁^Ser was isolated pure, while the CCA-half derived from tRNA₂^Ser was isolated as a mixture with the CCA-half from tRNA₁^Ser from which the terminal CpCpA had been cleaved off.The acceptor activity of the combined complementary half molecules was 90% of the one of intact tRNA^Ser. The Michaelis constant and maximal velocity of amino-acylation were found to be identical for tRNA^Ser and the combined fragments.When half molecules were present at different ratios in aminoacylation studies it was found that one pG-half molecule can mediate the charging of several CCA-half molecules. There are indications that the CCA-half molecule alone can accept some serine. The CCA-half molecule alone can be aminoacylated to a rather high degree in the presence of an excess of tRNA_ox^Ser or tRNA^Ser-a and to a small degree in the presence of tRNA_ox^Ala (yeast) but not at all in the presence of tRNA_ox^Phe or tRNA_ox^Val (E. coli).Combinations of half molecules from tRNA^Ser with the opposite half molecules from tRNA^Phe could not be aminoacylated with Ser or Phe or 15 other amino acids although one of the combinations was well associated according to gel electrophoresis and differential melting curves.     

A tRNA Val(GAC) gene of chloroplast origin in sunflower mitochondria is not transcribed

A tRNA^Val (GAC) gene is located in opposite orientation 552 nucleotides (nt) down-stream of the cytochrome oxidase subunit III (coxIII) gene in sunflower mitochondria. The comparison with the homologous chloroplast DNA revealed that the tRNA^Val gene is part of a 417 nucleotides DNA insertion of chloroplast origin in the mitochondrial genome. No tRNA^Val is encoded in monocot mitochondrial DNA (mtDNA), whereas two tRNA^Val species are coded for by potato mtDNA. The mitochondrial genomes of different plant species thus seem to encode unique sets of tRNAs and must thus be competent in importing the missing differing sets of tRNAs.