Tyrosine tRNA Research Articles

The Escherichia coli FIS protein is not required for the activation of tyrT transcription on entry into exponential growth.

The Escherichia coli DNA bending protein factor for inversion stimulation (FIS), is neither necessary nor responsible for the stimulation of transcription from the wild type promoter for the tyrT operon (encoding a species of tyrosine tRNA) that occurs upon resumption of exponential growth. This conclusion is unexpected given that the regulatory element required for optimal transcription of tyrT contains three binding sites for FIS protein. In addition, it is in apparent conflict with reports from other laboratories which have described FIS-dependent activation of the stable RNA promoters rrnB P1 and thrU(tufB) in vivo. However, tyrT transcription is stimulated in a FIS-dependent manner both in vivo and in vitro when promoter function is impaired by mutation of the promoter itself or by the addition of the polymerase effector guanosine 5'-diphosphate 3'-diphosphate. These conditions, which expose a requirement for activation of stable RNA synthesis by FIS, suggest that FIS serves an adaptive role permitting high levels of stable RNA transcription on nutritional shift-up when RNA polymerase levels are depleted. In principle such a mechanism could confer a significant selective advantage thus accounting for the conservation of FIS binding sites in the regulatory regions of stable RNA promoters.

Synthetase competition and tRNA context determine the in vivo identity of tRNA discriminator mutants

The discriminator nucleotide (position 73) in tRNA has long been thought to play a role in tRNA identity as it is the only variable single-stranded nucleotide that is found near the site of aminoacylation. For this reason, a complete mutagenic analysis of the discriminator in three Escherichia coli amber suppressor tRNA backgrounds was undertaken; supE and supE-G1C72 glutamine tRNAs, gluA glutamate tRNA and supF tyrosine tRNA. The effect of mutation of the discriminator base on the identity of these tRNAs in vivo was assayed by N-terminal protein sequencing of E. coli dihydrofolate reductase, which is the product of suppression by the mutated amber suppressors, and confirmed by amino acid specific suppression experiments. In addition, suppressor efficiency assays were used to estimate the efficiency of aminoacylation in vivo. Our results indicate that the supE glutamine tRNA context can tolerate multiple mutations (including mutation of the discriminator and first base-pair) and still remain predominantly glutamine-accepting. Discriminator mutants of gluA glutamate tRNA exhibit increased and altered specificity probably due to the reduced ability of other synthetases to compete with glutamyl-tRNA synthetase. In the course of these experiments, a glutamate-specific mutant amber suppressor, gluA-A73, was created. Finally, in the case of supF tyrosine tRNA, the discriminator is an important identity element with partial to complete loss of tyrosine specificity resulting from mutation at this position. It is clear from these experiments that it may not be possible to assign a specific role in tRNA identity to the discriminator. The identity of a tRNA in vivo is determined by competition among aminoacyl-tRNA synthetases, which is in turn modulated by the nucleotide substitution as well as the tRNA context.

Mapping the putative RNA helicase genes by sequence overlaping

An 'electronic' gene mapping procedure based on computer-aided search for overlapping gene sequences was used to identify adjacent genes and localize several putative RNA helicase genes to different chromosomes. PRP28 and AMD1 genes map to the right arm of chromosome IV next to sup2, which encodes a tyrosine tRNA. PRP16, previously mapped to chromosome XI, is tightly linked to MRP-L20. PRP22 is adjacent to PRE1, whose chromosomal location is currently unknown. The utility of this approach in yeast gene mapping is evaluated.

Mutants of Escherichia coli initiator tRNA that suppress amber codons in Saccharomyces cerevisiae and are aminoacylated with tyrosine by yeast extracts.

We recently described mutants of Escherichia coli initiator tRNA that suppress amber termination codons (UAG) in E. coli. These mutants have changes in the anticodon sequence (CAU----CUA) that allow them to read the amber codon and changes in the acceptor stem that allow them to bind to the ribosomal aminoacyl (A) site. We show here that a subset of these mutants suppress amber codons in Saccharomyces cerevisiae and that they are aminoacylated with tyrosine by yeast extracts. Analysis of a number of mutants as substrates for yeast tyrosyl-tRNA synthetase has led to identification of the C1.G72 base pair and the discriminator base A73, conserved in all eukaryotic cytoplasmic and archaebacterial tyrosine tRNAs, as being important for recognition. Our results suggest that the C1.G72 base pair and the discriminator base, in addition to the anticodon nucleotides previously identified [Bare, L.A. & Uhlenbeck, O.C. (1986) Biochemistry 25, 5825-5830] as important in yeast tyrosyl-tRNA synthetase recognition, may comprise the critical identity determinants in yeast tyrosine tRNA.

Mutations Induced by Ionizing Radiation in a Plasmid Replicated in Human Cells: I. Similar, Nonrandom Distribution of Mutations in Unirradiated and X-Irradiated DNA

The Escherichia coli supF gene encoding the suppressor tyrosine tRNA in a human shuttle plasmid, pZ189, was used as a target for molecular analysis of X-ray-induced mutations in human lymphoblastoid cells. Following replication of the in vitro-irradiated plasmid in human cells, the mutant supF-containing molecules were cloned by phenotypic screening in E. coli and the nature of the mutations was determined by direct sequencing of the tRNA gene. At 160 Gy the mutant frequency was 13 times (0.39%) that observed in unirradiated controls (0.031%). When control plasmid was replicated directly in E. coli, the mutant frequency was 16 times less than that of the plasmid passaged through the human cells. The distribution of mutations was highly nonrandom and remarkably similar in both irradiated and control DNAs. The majority of the mutations were transitions involving G.C pairs and occurred selectively at most 5'-TC (3'-AG) sequences. These mutations at C's were preferentially distributed in the nontranscribed strand. We propose that mutations in the control plasmid result from oxidative damages that occur during and/or after its incorporation into human cells and that these damages are similar to those induced by ionizing radiation. The hot spots for mutations suggest that the proximate nucleotide sequence and the overall conformation of the target DNA are important in the production and/or processing of these damages during repair and replication.

A Single Base Pair Dominates over the Novel Identity of an Escherichia coli Tyrosine tRNA in Saccharomyces cerevisiae

The Escherichia coli su+3 tyrosine tRNA was shown recently to be a leucine-specific tRNA in Saccharomyces cerevisiae. This finding raises the possibility that some determinants for tRNA identity in E. coli may be different in S. cerevisiae. To investigate whether the fungal system is sensitive to the major determinant for alanine acceptance in E. coli, a single G3 . U70 base pair was introduced into the acceptor helix of the su+3 tyrosine tRNA. This substitution converts the identity of the E. coli suppressor in S. cerevisiae from leucine to alanine. Thus, as in E. coli, G3 . U70 is a strong determinant for alanine acceptance that can dominate over other features in a tRNA that might be recognized by alternative charging enzymes.

A single base pair dominates over the novel identity of an Escherichia coli tyrosine tRNA in Saccharomyces cerevisiae.

The Escherichia coli su+3 tyrosine tRNA was shown recently to be a leucine-specific tRNA in Saccharomyces cerevisiae. This finding raises the possibility that some determinants for tRNA identity in E. coli may be different in S. cerevisiae. To investigate whether the fungal system is sensitive to the major determinant for alanine acceptance in E. coli, a single G3 . U70 base pair was introduced into the acceptor helix of the su+3 tyrosine tRNA. This substitution converts the identity of the E. coli suppressor in S. cerevisiae from leucine to alanine. Thus, as in E. coli, G3 . U70 is a strong determinant for alanine acceptance that can dominate over other features in a tRNA that might be recognized by alternative charging enzymes.

The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons

Expression of the RNA replicase domain of tobacco mosaic virus (TMV) and certain protein-coding regions in other plant viruses, is mediated by translational readthrough of a leaky UAG stop codon. It has been proposed that normal tobacco tyrosine tRNAs are able to read the UAG codon of TMV by non-conventional base-pairing but recent findings that stop codons can also be bypassed as a result of extended translocational shifts (tRNA hopping) have encouraged a re-examination. In light of the alternatives, we investigated the sequences flanking the leaky UAG codon using an in vivo assay in which bypass of the stop codon is coupled to the transient expression of β-glucuronidase (GUS) reporter genes in tobacco protoplasts. Analysis of GUS constructions in which codons flanking the stop were altered allowed definition of the minimal sequence required for readthrough as UAG-CAA-UUA. The effects of all possible single-base mutations in the codons flanking the stop indicated that 3′ contexts of the form CAR-YYA confer leakiness and that the 3′ context permits readthrough of UAA and UGA stop codons as well as UAG. Our studies demonstrate a major role for the 3′ context in the readthrough process and do not support a model in which the UAG is bypassed exclusively as a result of anticodon-codon interactions. No evidence for tRNA hopping was obtained. The 3′ context apparently represents a unique sequence element that affects translation termination.

An Escherichia coli tyrosine transfer RNA is a leucine-specific transfer RNA in the yeast Saccharomyces cerevisiae.

While the Escherichia coli Su-3 (tyrT) tyrosine tRNA suppressor inserts only tyrosine at amber codons in E. coli, we show here that in Saccharomyces cerevisiae this tRNA inserts leucine and no significant amounts of any other amino acid. Thus, the E. coli tyrosine tRNA is functionally a leucine tRNA in yeast cytoplasm. This functional identity may correlate with a structural relationship between the E. coli tyrosine and yeast leucine tRNAs, which are both members of the uncommon type II class of tRNA structures. The results raise the possibility that in evolution a tRNA may be more closely related to a tRNA of different acceptor specificity, but of the same type class, than to one with the same amino acid specificity, but of a different type class.

A gene encoding a tyrosine tRNA synthetase is located near Sacs in Bacillus subtilis

Within the frame of an attempt to sequence the whole Bacillus subtilis genome, a region of 5.5 kbp of the B. subtilis chromosome near the sacS locus has been sequenced. It contains five complete coding sequences, including the sequence of sacY, three unknown CDS and a sequence coding for a tyrosine tRNA synthetase. That the corresponding CDS encodes a functional synthetase has been demonstrated by complementation of an Escherichia coli mutant possessing a thermosensitive tRNA synthetase. Insertion of a kanamycin resistance cassette in the B. subtilis chromosome at the corresponding locus resulted, however, in no apparent phenotype, demonstrating that this synthetase is dispensable. Finally phylogenetic relationships between known tyrosine and tryptophan tRNA synthetases are discussed.

A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase.

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.

A Bacterial Amber Suppressor in Saccharomyces cerevisiae Is Selectively Recognized by a Bacterial Aminoacyl-tRNA Synthetase

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.

Structural basis for misaminoacylation by mutant E. coli glutaminyl-tRNA synthetase enzymes.

A single-site mutant of Escherichia coli glutaminyl-synthetase (D235N, GlnRS7) that incorrectly acylates in vivo the amber suppressor supF tyrosine transfer RNA (tRNA(Tyr] with glutamine has been described. Two additional mutant forms of the enzyme showing this misacylation property have now been isolated in vivo (D235G, GlnRS10; I129T, GlnRS15). All three mischarging mutant enzymes still retain a certain degree of tRNA specificity; in vivo they acylate supE glutaminyl tRNA (tRNA(Gln] and supF tRNA(Tyr) but not a number of other suppressor tRNA's. These genetic experiments define two positions in GlnRS where amino acid substitution results in a relaxed specificity of tRNA discrimination. The crystal structure of the GlnRS:tRNA(Gln) complex provides a structural basis for interpreting these data. In the wild-type enzyme Asp235 makes sequence-specific hydrogen bonds through its side chain carboxylate group with base pair G3.C70 in the minor groove of the acceptor stem of the tRNA. This observation implicates base pair 3.70 as one of the identity determinants of tRNA(Gln). Isoleucine 129 is positioned adjacent to the phosphate of nucleotide C74, which forms part of a hairpin structure adopted by the acceptor end of the complexed tRNA molecule. These results identify specific areas in the structure of the complex that are critical to accurate tRNA discrimination by GlnRS.

Oocyte and somatic tyrosine tRNA genes in Xenopus laevis.

Over a period of many months, Xenopus oocytes stockpile large quantities of tRNA for use during the first few hours of embryogenesis. To test the idea that these tRNAs are transcribed from one set of genes and that another set is used by somatic cells, we used synthetic oligonucleotides to analyze the sequence and steady-state levels of unspliced tyrosine tRNA precursors in Xenopus laevis oocytes, embryos, and cultured kidney cells. These analyses identify four kinds of tyrosine tRNA genes, two oocyte-type and two somatic-type, whose unspliced transcripts are distinguishable from one another by their different 5' leader and intervening sequences. The oocyte-type tyrosine tRNA precursors are present in oocytes, very abundant in gastrula embryos, but absent from postembryonic somatic cells. The somatic-type precursors are undetectable in oocytes but are found in gastrula and later stage embryos and in somatic cells. The major switch from oocyte-type to somatic-type transcripts occurs early during embryogenesis, between the midblastula transition and the onset of neurulation, but some oocyte-type precursors are also detectable in tadpoles.

Anti-mutagenic effect of ultraviolet light on spontaneous tyrosine tRNA ochre suppressor mutations in Escherichia coli.

The numbers of tyrosine tRNA ochre suppressor mutations arising spontaneously or after UV irradiation in different strains of Escherichia coli K12 are considered. The DNA sequence change requisite for this type of mutation would be a transversion at a cytosine between two purines, where pyrimidine-pyrimidine photoproducts could not form. We find that UV mutagenesis does not produce these tyrosine tRNA ochre suppressor mutations. With lexA51 recA441 defective cells, the spontaneous yield of these mutations is elevated and UV irradiation produces a significant decrease in the numbers of this particular mutation. As explanation we suggest that the spontaneous appearance of these mutations reflects mutation at apurinic sites, the efficiency of which is elevated in lexA51 recA441 cells (with derepressed SOS functions and an activated form of RecA protein). The addition of UV damage in the DNA of these cells cannot further stimulate the positive functions that are required for the production of these mutations and are typically associated with UV mutagenesis (induction of SOS functions, activation of RecA protein and introduction of a targeting photoproduct) but apparently can have a negative effect on mutagenesis, hitherto not realized.

Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs.

Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.

Introduction of an intervening sequence into a human serine suppressor tRNA gene: effects on gene expression in vitro and in vivo.

The 13 nucleotide Xenopus laevis tyrosine tRNA gene intervening sequence was into a human serine suppressor tRNA gene which lacked an intron, by site-directed mutagenesis. Analysis of the products of in vitro transcription in a HeLa cell extract indicates that the intervening sequence is accurately removed to generate a mature sized RNA identical to that obtained from an intron-less gene. Analysis of the transcripts obtained in vitro and in vivo shows that the U in the CUA anticodon sequence is partially modified to psi. Total TRNA isolated from cells infected with recombinant SV40 viruses carrying the mutant tRNA genes is active in suppression of UAG codons in a reticulocyte cell-free system. Cotransfection of COS cells with the mutant tRNA genes and a mutant chloramphenicol acetyltransferase gene containing the termination codon UAG demonstrated that the tRNA functions as a UAG suppressor in vivo. Analysis of 32P-labeled RNA obtained from infected cells showed, however, that cells infected with the intron-containing gene accumulate less mature tRNA than cells infected with the intron-less tRNA genes.

Isolation and characterization of conditional lethal amber nonsense mutants of vesicular stomatitis virus.

We describe the isolation and characterization of conditional lethal amber nonsense mutants of vesicular stomatitis virus (VSV), Indiana serotype. The mutants were isolated from a chemically mutagenized stock of wild-type virus by their ability to grow on genetically engineered cells which express a Xenopus laevis amber suppressor tyrosine tRNA gene (su+ cells) but not on the non-suppressor parental cells (su- cells). Five mutations were assigned to complementation group I (the L gene) and one to complementation group V (the G gene) by complementation analysis using temperature-sensitive mutants representing each of the five VSV cistrons. Four of the group I mutants were observed to synthesize a novel polypeptide species in su+ cells. Immunoprecipitation and immunoblotting studies using monospecific antisera directed against the N and C termini of the VSV L protein showed that the novel polypeptide species contain N terminal- but not C terminal-specific sequences and can thus be considered to be truncated versions of the L protein. In addition a protein which again contained N terminal- but not C terminal-specific sequences could be identified for the fifth group I mutant. Revertants of four of the group I mutants were isolated on the su- cells. The revertants all synthesized normal L protein but not the putative truncated version.

An E. coli aminoacyl-tRNA synthetase can substitute for yeast mitochondrial enzyme function in vivo

We have investigated the function of an E. coli aminoacyl-tRNA synthetase in S. cerevisiae strains that are respiration-deficient because of a mutation or a gene disruption in the nuclear encoded gene for the mitochondrial tyrosyl-tRNA synthetase. Although the yeast mitochondrial and E. coli tyrosine tRNAs differ significantly in sequence, expression of the E. coli tyrosyl-tRNA synthetase from a gene fusion restores respiration. The fusion gene contains a presumptive sequence for mitochondrial import from the mitochondrial tyrosyl-tRNA synthetase gene fused to the E. coli coding region. The fusion protein is incorporated into mitochondria. This incorporation and the rescue of the respiratory defect require the presumptive sequence for mitochondrial import. These experiments suggest a more limited definition of the identify of a tyrosine tRNA.

The chloroplast glutamate tRNA gene required for δ-aminolevulinate synthesis

The first major intermediate in the tetrapyrrole biosynthetic pathway is δ-aminolevulinate (δ-ALA). The synthesis of δ-ALA in greening barley requires an RNA (δ-ALA-RNA) which is aminoacylated with glutamate by a tRNA ligase. δ-ALA-RNA isolated from barley plastids and a synthetic nucleotide oligomer were used as hybridization probes to isolate the gene encoding δ-ALA-RNA from a collection of barley DNA chloroplast clones. Sequence of the barley δ-ALA-RNA reveals a typical tRNA sequence which is identical to the wheat glutamate tRNA gene. The tyrosine and aspartate tRNA genes are closely linked to δ-ALA-RNA on a 1.6 kb HindIII-EcoRI restriction fragment. The glutamate tRNA gene is transcribed in the dark and in the light under all the conditions tested. S1 mapping was used to locate the potential regulatory sequences and to establish that the three tRNAs are processed from a single precursor RNA. A single gene encodes the δ-ALA-RNA which functions in δ-ALA synthesis and the glutamate tRNA which functions in chloroplast protein synthesis. The two RNA products may differ in posttranscriptional modifications.