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PubMed1969

Transfer RNA. 3. Reconstitution of alanine acceptor activity from fragments produced by specific cleavage of tRNA Ala-II at its anticodon.

This report describes experiments showing that tRNA(Ala) (II) can be cleaved specifically at the G residue of its anticodon to give "half" molecules in high yield. Neither of these fragments has alanine-acceptor activity, but this activity can be reconstituted by mixing the fragments in the presence of Mg(+2). In dilute salt, on the other hand, the active complex dissociates spontaneously at 25 degrees into the two fragments. Thus, both "halves" of tRNA(Ala) (II) are necessary for acceptor activity and, in the presence of Mg(+2), combination of these fragments to give an active duplex is thermodynamically favorable.

Alanine↗
PubMed1994

Evolution of the WANCY region in amniote mitochondrial DNA.

In most vertebrate mitochondrial genomes, the site for initiation of light-strand replication, OL, is found within a cluster of five transfer RNA (tRNA) genes (tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), and tRNA(Tyr)). This region and part of the adjacent cytochrome c oxydase subunit I (COI) gene were sequenced for two crocodilian, two turtle, and one snake species and for Sphenodon punctatus; part of the adjacent nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2) gene was also sequenced for the crocodilian and turtle species. All had the typical vertebrate gene order. The turtles and the snake have a lengthy noncoding sequence between the tRNA(Asn) and tRNA(Cys) genes that we assumed to be homologous to the mammalian OL. The crocodilians and Sphenodon lack such a sequence, a condition they share with birds. Most proposed phylogenies for the amniotes require that OL at this position was lost at least twice during their diversification or was evolved independently more than once. Within the five tRNA genes, frequencies of substitutions are much higher in loops than in stems. Many loops vary dramatically in size among the species; in the most extreme case, the D-arm of the Sphenodon tRNA(Cys) is a "D-arm replacement" loop of seven nucleotides. Frequency of transitions in stems is relatively uniform across tRNAs, but frequency of transversions varies greatly. Mismatches in stems are infrequent, and their relative frequency in a specific tRNA is unrelated to the frequency of substitution in the corresponding gene. Several features of mammalian mitochondrial tRNAs are conserved in WANCY tRNAs throughout amniotes. The inferred initiation codon for COI is GTG in crocodilians, turtles, and the snake, a condition they share with fishes, certain amphibians, and birds. TTG appears to be the initiation codon for COI in Sphenodon; if correct, this would be a novel initiation codon for vertebrate mitochondrial DNA. Phylogenetic analyses of the inferred amino acid sequences of ND2 and COI support the sister-group relationship of birds and crocodilians and suggest that mammals are an early derived lineage within the amniotes.

Amino Acid Sequence↗
PubMed1991

delta-Aminolevulinic Acid Biosynthesis from Glutamatein Euglena gracilis: Photocontrol of Enzyme Levels in a Chlorophyll-Free Mutant.

Wild-type Euglena gracillis cells synthesize the key chlorophyll precursor, delta-aminolevulinic acid (ALA), from glutamate in their plastids. The synthesis requires transfer RNA(Glu) (tRNA(Glu)) and the three enzymes, glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase. Non-greening mutant Euglena strain W(14)ZNaIL does not synthesize ALA from glutamate and is devoid of the required tRNA(Glu). Other cellular tRNA(Glu)s present in the mutant cells were capable of being charged with glutamate, but the resulting glutamyl-tRNAs did not support ALA synthesis. Surprisingly, the mutant cells contain all three of the enzymes, and their cell extracts can convert glutamate to ALA when supplemented with tRNA(Glu) obtained from wild-type cells. Activity levels of the three enzymes were measured in extracts of cells grown under a number of light conditions. All three activities were diminished in extracts of cells grown in complete darkness, and full induction of activity required 72 hours of growth in the light. A light intensity of 4 microeinsteins per square meter per second was sufficient for full induction. Blue light was as effective as white light, but red light was ineffective, in inducing extractable enzyme activity above that of cells grown in complete darkness, indicating that the light control operates via the nonchloroplast blue light receptor in the mutant cells. Of the three enzyme activities, the one that is most acutely affected by light is glutamate-1-semialdehyde aminotransferase, as has been previously shown for wild-type Euglena cells. These results indicate that the enzymes required for ALA synthesis from glutamate are present in an active form in the nongreening mutant cells, even though they cannot participate in ALA formation in these cells because of the absence of the required tRNA(Glu), and that the activity of all three enzymes is regulated by light. Because the absence of plastid tRNA(Glu) precludes the synthesis of proteins within the plastids, the three enzymes must be synthesized in the cytoplasm and their genes encoded in the nucleus in Euglena.

Journal Article↗
PubMed2000

Two Tandemly Arrayed Transfer-RNA-Derived SINEs of the Medaka (Oryzias latipes).

Two tandemly arrayed short interspersed repetitive element (SINE) sequences were found in medaka (Oryzias latipes). These two SINE sequences, designated SINE1 and SINE2, were flanked by a 180-bp AT-rich region. Both appeared to be derived from transfer RNA. The former exhibited 80% sequence homology to human tRNA(Ala) and the latter exhibited 94% sequence homology to rat tRNA(Ser). SINE1 contained the retroviral U5 region, whereas SINE2 did not. This is the first sequence-level demonstration of the existence of neighboring SINEs in medaka.

Journal Article↗
PubMed2002

BRUCE: a program for the detection of transfer-messenger RNA genes in nucleotide sequences.

A computer program, BRUCE, was developed for the identification of transfer-messenger RNA (tmRNA) genes. The program employs heuristic algorithms to search for a tRNA(Ala)-like secondary structure surrounding a short sequence encoding the tag peptide. In the 57 completely sequenced bacterial genomes where tmRNA genes have been reported previously, BRUCE identified all with no false positives. In addition, BRUCE found 99 of the 100 tmRNAs identified previously in other bacteria, red chloroplasts and cyanelles. The output of the program reports the proposed tRNA secondary structure, the tmRNA gene sequence and the tag peptide.

Algorithms↗
PubMed1971

The yeast phenylalanyl-transfer RNA synthetase recognition site: the region adjacent to the dihydrouridine loop.

Purified yeast phenylalanyl-tRNA synthetase can aminoacylate (yeast) tRNA(Phe), (wheat) tRNA(Phe), and (Escherichia coli) tRNA(1) (Val) (1, 2). We now report that this synthetase can also aminoacylate (E. coli) tRNA(Phe) and (E. coli) tRNA(1) (Ala). Highly purified (E. coli) tRNA(Phe) is heterologously aminoacylated to approximately 90% of the extent achieved with the homologous enzyme (crude E. coli phenylalanyl-tRNA synthetase). Pure (E. coli) tRNA(1) (Ala) (the major species) is heterologously aminoacylated to 70% of the extent achieved with the homologous synthetase (crude E. coli alanyl-tRNA synthetase).(E. coli) tRNA(Phe) is the fourth purified transfer RNA of known sequence to be shown to be an acceptable substrate for purified yeast phenylalanyl-tRNA synthetase. A comparison of these sequences shows that only one region is extremely similar in all four tRNAs. This region is located adjacent to the dihydrouridine loop, and consists of the nucleotides [Formula: see text] We conclude that this is the synthetase recognition site for yeast phenylalanyl-tRNA synthetase. This conclusion is further supported by partial fragment analysis of (E. coli) tRNA(1) (Ala).

Binding Sites↗
PubMed1992

Glutamyl-transfer RNA: a precursor of heme and chlorophyll biosynthesis.

In green plants, archaebacteria and many eubacteria, the porphyrin ring that is common to both chlorophyll and heme is synthesized from 5-aminolevulinic acid (ALA) via an interesting pathway. This two-step process involves the unusual enzymes glutamyl-tRNA reductase and glutamate-1-semialdehyde 2,1-aminomutase. Interest in this pathway has increased since it was discovered that a tRNA cofactor was required for the formation of ALA. This tRNA(Glu) is common to the biosyntheses of both porphyrins and proteins.

Aldehyde Oxidoreductases↗
PubMed1993

Alteration of transfer ribonucleic acid of Neisseria meningitidis during growth in the presence of human transferrin.

Iron-related tRNA alterations have been shown to occur in several pathogens but nothing has been reported about the effect of iron on meningococcal tRNA. The chromatographic elution profile of 3H-tryptophan-tRNA(trp) from a Neisseria meningitidis strain grown under different conditions was examined. The profile showed an early (P1) and a late (P2) eluting species of tRNA(trp), but the proportion of the two species varied under different growth conditions. The elution profile of trp-tRNA(trp) from bacteria grown in iron-sufficient Mueller Hinton broth yielded a minor P1 species and a major P2 species, whereas under iron-restricted growth induced by desferrioxamine, the pattern was one of a major P1 species and minor P2 species. Iron-restriction induced by human transferrin (HTF) resulted in almost equal amounts of the two tRNA(trp) species (P1 congruent to P2). Differences in the proportions of the tRNA species were also found between cells grown in liquid medium (P1 << P2) and on the same medium solidified with agar (P1 >> P2). The growth phase of the bacteria did not have any effect on the tRNA(trp) elution profile. Changes in tRNA(trp) induced by HTF were readily and completely reversible when the cells were transferred to an iron-rich medium, but those induced by desferrioxamine remained irreversible for a long period (16 h) after such transfer.

Culture Media↗
PubMed1976

Deletion mapping of mitochondrial transfer RNA genes in Saccharomyces cerevisiae by means of cytoplasmic petite mutants.

Mitochondrial transfer RNA genes have been ordered relative to the position of five mitochondrial drug resistance markers, namely, chloramphenicol (C),1 erythromycin (E), oligomycin I and II (OI, OII), and paromomycin (P). Forty-six petite yeast clones that were genetically characterized with respect to these markers were used for a study of these relationships. Different regions of the mitochondrial genome are deleted in these individual mutants, resulting in variable loss of genetic markers. Mitochondrial DNA was isolated from each mutant strain and hybridized with eleven individual mitochondrial transfer RNAs. The following results were obtained: i) Of the seven petite clones that retained C, E, and P resistance markers (but not O1 or O11), four carried all eleven transfer RNA genes examined; the other three clones lost several transfer RNA genes, probably by secondary internal deletion; ii) Prolyl and valyl transfer RNA genes were located close to the P marker, whereas the histidyl transfer RNA gene was close to the C marker; iii) Except for a glutamyl transfer RNA gene that was loosely associated with the O1 region, no other transfer RNA genes were found in petite clones retaining only the O1 and/or the OII markers; and iv) Two distinct mitochondrial genes were found for glutamyl transfer RNA, they were not homologous in DNA sequence and were located at two separate loci. The data indicate that the petite mitochondrial genome is the result of a primary deletion followed by successive additional deletions. Thus an unequivocal gene arrangement cannot be readily established by deletion mapping with petite mutants alone. Nevertheless, we have derived a tentative circular map of the yeast mitochondrial genome from the data; the map indicates that all but one of the transfer RNA genes are found between the C and P markers without forming a tight cluster. The following arrangement is suggested: -P-pro-val-ile-(phe, ala, tyr, asp)-glu2- (lys-leu)-his-C-E-O1-glu1-OII-P-.

Chloramphenicol↗
PubMed2001

Human tyrosine tRNA is also internally cleavable by E. coli ribonuclease P RNA ribozyme in vitro.

Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure. Previously, we experimentally showed that some Drosophila tRNA (tRNA(Ala), tRNA(His), and tRNA(iMet)) molecules fit to form another, non-cloverleaf, structure in which the 3'-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P). Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs. This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E. coli RNase P RNA. This tRNA is the first example for hyperprocessed non-Drosophila tRNAs. The results suggest that the hyperprocessing reaction can be a useful tool detect destablized tRNA molecules from any species.

Base Sequence↗
PubMed1995

Minor groove recognition of the conserved G.U pair at the Tetrahymena ribozyme reaction site.

The guanine-uracil (G.U) base pair that helps to define the 5'-splice site of group I introns is phylogenetically highly conserved. In such a wobble base pair, G makes two hydrogen bonds with U in a geometry shifted from that of a canonical Watson-Crick pair. The contribution made by individual functional groups of the G.U pair in the context of the Tetrahymena ribozyme was examined by replacement of the G.U pair with synthetic base pairs that maintain a wobble configuration, but that systematically alter functional groups in the major and minor grooves of the duplex. The substitutions demonstrate that the exocyclic amine of G, when presented on the minor groove surface by the wobble base pair conformation, contributes substantially (2 kilocalories.mole-1) to binding by making a tertiary interaction with the ribozyme active site. It contributes additionally to transition state stabilization. The ribozyme active site also makes tertiary contacts with a tripod of 2'-hydroxyls on the minor groove surface of the splice site helix. This suggests that the ribozyme binds the duplex primarily in the minor groove. The alanyl aminoacyl transfer RNA (tRNA) synthetase recognizes the exocyclic amine of an invariant G.U pair and contacts a similar array of 2'-hydroxyls when binding the tRNA(Ala) acceptor stem, providing an unanticipated parallel between protein-RNA and RNA-RNA interactions.

Animals↗
PubMed1985

Cloning and nucleotide sequence analysis of transfer RNA genes from Mycoplasma mycoides.

As part of an investigation of the tRNA genes of Mycoplasma mycoides, two HindIII fragments of mycoplasma DNA comprising 0.4 and 2.5 kilobases (kb), respectively, were cloned in pBR322 and their nucleotide sequences determined. Only one tRNA gene was found in the 0.4 kb fragment, the gene for tRNAArg with the anticodon TCT, while the 2.5 kb fragment contained nine different tRNA genes arranged in a cluster which presumably constitutes a transcriptional unit. The clustered tRNA genes, with their respective anticodons, were as follows: Arg (ACG), Pro (TGG), Ala (TGC), Met (CAT), Ile (CAT), Ser (TGA), fMet (CAT), Asp (GTC), and Phe (GAA).

Base Sequence↗
PubMed1993

A continuous spectrophotometric assay for the aminoacylation of transfer RNA by alanyl-transfer RNA synthetase.

A continuous spectrophotometric assay is described for the aminoacylation reaction catalyzed by Escherichia coli alanyl-transfer RNA synthetase. The assay is based on coupling the alanyl-tRNA synthetase-dependent formation of AMP to the lactate dehydrogenase oxidation of NADH. Oxidation of NADH, as monitored at 340 nm, is shown to be stoichiometric with the formation of alanyl-tRNA(Ala). This assay will facilitate the rapid accumulation and analysis of kinetic data for alanyl-tRNA synthetase and should be applicable to aminoacyl-tRNA synthetases in general.

Acylation↗
PubMed1995

Mutational analysis of vaccinia DNA ligase defines residues essential for covalent catalysis.

DNA ligation entails AMP transfer from ATP to the 5' end of DNA to form a DNA-adenylate structure, A(5')pp(5')N. A similar reaction involving GMP transfer occurs during 5' capping of eukaryotic mRNA. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs. The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase. Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps. We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities. Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase-AMP formation. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a common structural basis for covalent nucleotidyl transfer.

Amino Acid Sequence↗
PubMed1987

Polypeptide sequences essential for RNA recognition by an enzyme.

Many RNAs are complex, globular molecules formed from elements of secondary and tertiary structure analogous to those found in proteins. Little is known about recognition of RNAs by proteins. In the case of transfer RNAs (tRNAs), considerable evidence suggests that elements dispersed in both the one- and three-dimensional structure are important for recognition by aminoacyl tRNA synthetases. Fragments of alanine tRNA synthetase were created by in vitro manipulations of the cloned alaS gene and examined for their interaction with alanine-specific tRNA. Sequences essential for recognition were located near the middle of the polypeptide, juxtaposed to the carboxyl-terminal side of the domain for aminoacyl adenylate synthesis. The most essential part of the tRNA interaction strength and specificity was dependent on a sequence of fewer than 100 amino acids. Within this sequence, and in the context of the proper conformation, a segment of no more than 17 amino acids was responsible for 25% or more of the total synthetase-tRNA free energy of association. The results raise the possibility that an important part of specific RNA recognition by an aminoacyl tRNA synthetase involves a polypeptide segment that is short relative to the total size of the protein.

Adenosine Triphosphate↗
PubMed1983

Structure and organization of a cluster of sic tRNA genes in the space between tandem ribosomal RNA gene sets in Bacillus subtilis.

Hybridization of Southern blots of EcoRI digests of total Bacillus subtilis DNA with ribosomal RNA and transfer RNA probes provides evidence of highly clustered tRNA and rRNA genes, with several tRNA clusters being located in spaces which span between tandem ribosomal RNA gene sets. Clones containing these tRNA clusters were isolated from a Charon 4A library. One of them, denoted trrnB, was partially sequenced. A cluster of six tRNA genes was found, with anticodon assignments of Asn, Thr, Gly, Arg, Pro, Ala. This cluster is closely flanked on both sides by ribosomal RNA gene sets, which were identified by sequencing upward through the 5 S rRNA gene, across a space, and into the 23 S rRNA gene, and also sequencing downward into the 16 S rRNA gene. The tRNA gene cluster appears to be organized into at least two transcriptional units separated by an attenuator region. These transcriptional units may be components of the flanking ribosomal RNA operons. The putative promoter region of the downstream 16 S rRNA is organized differently from Escherichia coli; it is smaller and seems less complex. This gene organization provides insight into possible mechanisms for coordinate and differential control of transfer and ribosomal RNA gene expression.

Anticodon↗