tRNATyr Gene Research Articles

Plant nonsense suppressor tRNATyrgenes are expressed at very low levesin vitrodue to inefficient splicing of the intron-containing pre-tRNAS

Oligonucleotide-directed mutagenesis was used to generate amber, ochre and opal suppressors from cloned Arabidopsis and Nicotiana tRNA(Tyr) genes. The nonsense suppressor tRNA(Tyr) genes were efficiently transcribed in HeLa and yeast nuclear extracts, however, intron excision from all mutant pre-tRNAs(Tyr) was severely impaired in the homologous wheat germ extract as well as in the yeast in vitro splicing system. The change of one nucleotide in the anticodon of suppressor pre-tRNAs leads to a distortion of the potential intron-anticodon interaction. In order to demonstrate that this caused the reduced splicing efficiency, we created a point mutation in the intron of Arabidopsis tRNA(Tyr) which affected the interaction with the wild-type anticodon. As expected, the resulting pre-tRNA was also inefficiently spliced. Another mutation in the intron, which restored the base-pairing between the amber anticodon and the intron of pre-tRNA(Tyr), resulted in an excellent substrate for wheat germ splicing endonuclease. This type of amber suppressor tRNA(Tyr) gene which yields high levels of mature tRNA(Tyr) should be useful for studying suppression in higher plants.

Genes for tRNAGly, tRNAHis, tRNALys, tRNAPhe, tRNASer and tRNATyr are encoded in Oenothera mitochondrial DNA

The genes coding for tRNA(Gly), tRNA(His), tRNA(Lys), tRNA(Phe), tRNA(Ser) and tRNA(Tyr) have been identified in Oenothera mitochondrial DNA. Sequence analysis of these genes and their surrounding sequences are presented and compared with other known tRNA genes from plant mitochondria. All six deduced tRNA sequences can be folded into the classical cloverleaf structure model. Only the tRNA(His) gene shows high homology with the corresponding chloroplast gene and thus appears to be derived from a transfer event of chloroplast sequences into the mitochondrial genome. The sequences surrounding this gene, however, show little similarity with the chloroplast genome. The other five deduced tRNAs display a much lower similarity with their chloroplast counterparts and thus appear to be genuine mitochondrial tRNAs. These tRNAs are highly conserved between monocots and dicots with maximally three nucleotides differing between the Oenothera sequences and their wheat homologues. A purine-rich sequence is found upstream of each tRNA gene in Oenothera, similar to wheat mitochondrial tRNA genes, that could be involved in transcription signalling.

A yeast tRNA precursor containing a pre-mRNA intron is spliced via the pre-mRNA splicing mechanism.

We have replaced the 14 nucleotide long intervening sequence of the Saccharomyces cerevisiae SUP6 (ochre) tRNA(Tyr) gene by the 52 nucleotide long second intron of the S. cerevisiae MATa1 gene. Yeast cells containing this modified pre-tRNA showed the typical suppressor phenotype indicating that the MATa1 pre-mRNA intron was exactly excised in vivo from the primary tRNA transcript and a mature and functional tRNA was formed. Several lines of evidence show that the splicing reaction proceeded via the pre-mRNA splicing mechanism: the reaction yielded a lariat shaped excised intron with a lariat shaped intron-exon 2 molecule as intermediate; point mutations in the conserved UAC-UAAC box of the intron impaired splicing of the precursor RNA; in a temperature sensitive rna2 strain splicing of this tRNA precursor was inhibited at the restrictive temperature. Our results imply that in yeast the excision of a pre-mRNA intron is not dependent on the transcription apparatus by which it was generated and that transcription and splicing are uncoupled processes in vivo, too. Furthermore these data demonstrate that recognition of an RNA as a substrate for a pre-mRNA splicing reaction is, at least qualitatively, only intron dependent.

S. cerevisiae TFIIIB is the transcription initiation factor proper of RNA polymerase III, while TFIIIA and TFIIIC are assembly factors

The S. cerevisiae RNA polymerase III (pol III) transcription factor TFIIIB binds to DNA upstream of the transcription start site of the SUP4 tRNATyr gene in a TFIIIC-dependent reaction and to the major 5S rRNA gene in a reaction requiring TFIIIC and TFIIIA. It is shown here that TFIIIB alone correctly positions pol III for repeated cycles of transcription on both genes, with the same efficiency as fully assembled transcription complexes. Thus, TFIIIB is the sole transcription initiation factor of S. cerevisiae pol III; TFIIIC and TFIIIA are assembly factors for TFIIIB. The TFIIIB-dependent binding of pol 111 to the SUP4 tRNA and 5S rRNA genes has been analyzed in binary (protein and DNA only) and precisely arrested ternary (protein, DNA, and RNA) transcription complexes. Pol III unwinds at least 14 by of DNA at the SUP4 transcription start in a temperature-dependent process. The unwound DNA segment moves downstream with nascent RNA as a transcription bubble of approximately the same size.

Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster.

Drosophila melanogaster strains with a stably incorporated amber suppressor tRNA gene have been generated. A tRNATyr gene was site specifically mutated to produce an anticodon sequence that recognizes the amber codon and then introduced into Drosophila by using P-element-mediated transformation. Transformants from four integration events were recovered. Two integrations resulted in both male and female sterility, whereas the other two resulted in male sterility but female fertility. Strains derived from the two female-fertile integration events were shown to have a low level of amber-suppressing activity by their ability to suppress an amber mutation in a chloramphenicol acetyltransferase gene.

Three mitochondrial tRNA genes fromArabidopsis thaliana: evidence for the conversion of a tRNAPhegene into a tRNATyrgene

Three tRNA genes have been isolated from a genomic library of Arabidopsis thaliana: a tRNASer (GCU), a tRNATyr (GUA) and a tRNAGlu (UUC) genes. These genes are located closely on the same DNA fragment. The tRNASer and the tRNAGlu genes have both 99% sequence similarity with their mitochondrial counterparts from higher plants indicating that these three tRNA genes are mitochondrial. The tRNATyr gene shows a particular high sequence similarity with the mitochondrial tRNAPhe pseudogene from maize, and both genes are flanked by a tRNASer gene in the upstream region. Extensive sequence comparisons of the Arabidopsis thaliana mitochondrial sequence containing the three tRNA genes and the corresponding region from maize and soybean mitochondria have shown evidence that the tRNA Tyr gene has been generated from a mitochondrial tRNAPhe gene. The conversion was accomplished by three genetic events: a 4 base-pair deletion, a mutation and a recombination, which led to the transformation of the acceptor stem and the anticodon.

Wheat germ splicing endonuclease is highly specific for plant pre-tRNAs.

Intron-containing pre-tRNAs from organisms as different as yeast, Nicotiana, Xenopus and man are efficiently spliced and processed in a HeLa cell extract. They are also correctly processed in a wheat germ extract; however, the intron is removed only from the tobacco pre-tRNA. To determine whether plant pre-tRNA introns have any specific structural and/or sequence feature we have cloned two intron-containing tRNATyr genes from the plant Arabidopsis. Comparison of these genes, of the Nicotiana tRNATyr gene and of a Glycine max tRNAMet gene reveals that plant introns from three different species have no sequence homology and are only 11 to 13 nucleotides long. Thus, short length may be one important feature of plant introns. Furthermore, the 5' and 3' splice sites are separated by 4 bp in the extended anticodon stems of these pre-tRNA structures. In contrast, yeast and vertebrate introns are rather variable in length and the splice sites are separated by 5 or 6 bp. These differences in distance and relative helical orientation of the splice sites in plant pre-tRNAs versus pre-tRNAs from other organisms are obviously tolerated by the vertebrate splicing endonuclease, but not at all by the plant enzyme.

All human tRNATyr genes contain introns as a prerequisite for pseudouridine biosynthesis in the anticodon.

Two synthetic oligonucleotides, one specific for the 5' exon, the other spanning the splice junction, were used to show that (a) the human haploid genome contains at least 12 independent gene loci for tRNATyr, and (b) that all of them carry an intron. From one of the cloned human tRNATyr genes (pHtT1) the 20 bp intron was deleted to generate pHtT1 delta. Homologous in vitro transcription, fingerprint analyses of the products and elucidation of their nucleoside composition revealed that the pseudouridine (psi 35) in the center of the anticodon of tRNATyr was synthesized in the intron-containing precursor whereas this U to psi modification did not take place in precursors or mature tRNATyr derived from pHtT1 delta. On the basis of these results and of studies from other laboratories we suggest that the evolutionary pressure for maintaining introns in eukaryotic tRNAsTyr is this strict intron-requirement for psi 35 synthesis. Taking into account that all eukaryotic cytoplasmic tRNAsTyr contain a psi 35 we discuss here a special need for this modified nucleoside in stabilizing codon-anticodon interactions involving (a) classical base pairing upon translation of tyrosine codons and (b) unconventional interactions during UAG amber codon suppression by tRNATyrG psi A in eukaryotic cells.

A single base change in the extra-arm of yeast mitochondrial tyrosine tRNA affects its conformational stability and impairs aminoacylation.

The mitochondrial temperature-sensitive mutation tsm-8 maps on a 1.8 kb HpaII fragment of mitochondrial DNA (mt DNA) which contains genes for tRNA(Ala), tRNA(Ile) and tRNA(Tyr). The phenotype of this mutation is, among multiple pleiotropic defects, a temperature-induced reduction of mitochondrial translation. DNA sequencing of the HpaII fragment from the wild type and mutant tsm-8 revealed a single transversion from T to A in position 56 of the mutant tRNA(Tyr) gene. This nucleotide change disrupts a base pairing in the long extra arm of the tRNA cloverleaf. Revertants of the tsm-8 mutant restore correct base pairing in the extra arm by a second-site mutation in the tRNA(Tyr) gene. Analysis of the tRNA(Tyr) transcripts revealed that neither transcription nor processing of the tRNA is affected in the mutant. However, the base alteration destabilizes the conformation of the tRNA and affects its charging parameters. At the non-permissive temperature, the Michaelis-Menten constant of the mitochondrial tyrosyl-tRNA synthetase for the mutant tRNA is increased over 20-fold when compared to the wild-type tRNA. As a consequence, mitochondrial protein synthesis is drastically reduced at the restrictive temperature. Moreover, synthesis of apocytochrome b and of cytochrome oxidase subunit 3 is decreased relative to the other mitochondrially synthesized polypeptides.

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.

Effects of alterations in the 3' flanking sequence on in vivo and in vitro expression of the yeast SUP4-o tRNATyr gene.

The SUP4-o gene of Saccharomyces cerevisiae codes for an altered tRNATyr capable of suppressing ochre mutations. We constructed mutant SUP4-o genes with deletions in the 3'-flanking sequence and tested each for its ability to suppress ochre mutations in transformed yeast cells. The effects of the different 3' deletions on various aspects of in vitro transcription and RNA processing were also determined, using a yeast cell-free extract. Deletions that leave five or fewer consecutive T residues in the 3'-flanking sequence of SUP4-o were found to result in decreased efficiency of transcription termination, both in vitro and in vivo. Unexpectedly, the suppression strength of each mutant SUP4-o gene is highly correlated with the relative extent of transcription termination at the 3' end of the gene. This result indicates that SUP4-o readthrough transcripts are not efficiently processed to functional suppressor tRNA in yeast cells. Deletions that extend into the T cluster in the 3'-flanking sequence also significantly decrease the ability of SUP4-o to compete for a transcription factor that is limiting in our extracts. This latter finding implies that the 3'-flanking sequence of SUP4 plays a role in transcription factor binding.

First identification of an amber nonsense mutation in Schizosaccharomyces pombe: major differences in the efficiency of homologous versus heterologous yeast suppressor tRNA genes

In Saccharomyces cerevisiae ochre and opal, as well as amber mutations are known, whereas in the fission yeast Schizosaccharomyces pombe no amber alleles have been described. We have characterized trp1-566, an amber allele in the trp1 locus of S. pombe. The identification of trp1-566 as an amber allele is based on the following results: (a) The nonsense allele can be converted to an ochre allele by nitrosoguanidine mutagenesis. (b) trp1-566 is suppressed by a bona fide S. pombe amber suppressor tRNA, supSI. The supSI gene was obtained by primer-directed in vitro mutagenesis of a tRNASer from S. pombe. Unexpectedly, an S. cerevisiae amber suppressor tRNASer, supR21, transformed into S. pombe, failed to suppress trp1-566. Northern analysis of S. pombe transformants, containing supRL1 or S. cerevisiae tRNALeu or tRNATyr genes reveals that these genes are not transcribed in the fission yeast. As an additional tool for the analysis of nonsense mutations in S. pombe, we obtained by nitrosoguanidine mutagenesis two unlinked amber suppressor alleles, sup13 and sup14, which act on trp1-566.

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).

A test for intron function in the yeast actin gene.

Many eukaryotic genes contain intervening sequences (IVS), but the rationale for their existence remains a mystery. Previous studies done in our laboratory demonstrated that the intron in a yeast tRNATyr gene, SUP6, does have a function. We used the same approach to determine the role of introns in nuclear genes encoding messenger RNAs. A single actin gene with one intron exists in Saccharomyces cerevisiae. The level of actin in yeast appears to be crucial to viability: either too much or too little actin inhibits growth. Therefore, small effects on synthesis of actin protein resulting from the removal of the actin gene intron would be expected to cause measurable changes in cell growth. In the present study, an intron-deleted actin gene was constructed in vitro and was used to replace the single resident actin gene in a haploid strain. Analysis of the cells carrying the intron-deleted actin gene shows that the intervening sequence is not essential for actin gene expression.

Structure and cotranscription of tobacco chloroplast genes for tRNAGlu(UUC), tRNATyr(GUA) and tRNAAsp(GUC).

The location and nucleotide sequences of tobacco chloroplast genes for tRNAGlu(UUC), tRNATyr(GUA) and tRNAAsp(GUC) have been determined. These genes lie midway between the genes for alpha and beta/epsilon subunits of H+-ATPase on the large single-copy region of the chloroplast DNA. The gene organization is tRNAGlu - 59bp spacer - tRNATyr - 108bp spacer - tRNAAsp on the same DNA strand. Northern blot hybridization studies revealed that these three tRNA genes are cotranscribed. The transcription initiation site was localized at 24 bp upstream from the tRNAGlu coding region and its termination site at 90 bp downstream from the tRNAAsp coding region by S1 mapping. The tricistronic tRNA precursor is thus calculated to be 512 bases long. Its processing was also studied by S1 mapping.

Structural features of yeast tRNA genes which affect transcription factor binding.

Transcription of yeast tRNA genes in vitro requires, in addition to RNA polymerase III, two accessory factors which are resolved by ion-exchange chromatography. One of these transcription factors (factor C) binds to tRNA genes. The stability of factor C-tRNA gene complexes is gene-dependent: the tRNAAGGArg gene forms a highly stable complex while tRNA3Leu and tRNATyr gene complexes are unstable under our standard assay conditions. To determine how differences in tRNA gene structure affect factor C binding, mutant tRNATyr genes, internally deleted tRNA3Leu genes and hybrid transcription units containing both tRNATyr and tRNA3Leu segments were compared in their abilities to stably bind factor C. Sequence changes in either of the two highly conserved promoter elements (A block and B block) affect factor C complex stability. Changes towards the consensus sequence increase complex stability while changes away from the consensus sequence drastically reduce stability. Also, the distance separating the A and B blocks affects complex stability; 34-53 bp gives highest stability. These results indicate that the stable binding of transcription factor C to tRNA genes involves interactions with both A block and B block sequences.

Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene.

Deletion mutations ending in the 5'-flanking sequences of the Saccharomyces cerevisiae SUP4-o gene have been analyzed for their effects on gene expression. This ochre-suppressing tRNATyr gene was cloned into a S. cerevisiae centromeric plasmid, and its level of in vivo expression was monitored by observing the suppressor phenotype of the gene after transformation into S. cerevisiae. A deletion mutant that retains only four base pairs of the 5'-flanking sequence is profoundly deficient in expression; deletion mutants extending to positions -18, -17, -16, or -15 are moderately deficient; deletion mutants extending to positions -36 or -27 are slightly defective; and mutants retaining more than 60 base pairs of the original 5'-flanking DNA are expressed normally. In some cases, the cloning procedure led to the introduction of multiple BamHI linkers at the SUP4-o-vector fusion site, and in one instance, the resulting structure dramatically affects gene function: the presence of three linkers abutting a -18 deletion completely inhibits the in vivo expression of SUP4-o. In contrast, three linkers that abut a -77 deletion have no effect on in vivo expression. The template properties of these plasmids in a homologous in vitro transcription system parallel the levels of in vivo expression, suggesting that the mutations predominantly affect transcription. The data demonstrate that there are significant functional constraints on the 5'-flanking sequences of this RNA polymerase III-transcribed gene. The dramatic effects of the multiple linker insertion at position -18 suggest that there may be extensive melting of the DNA in this region during normal transcription initiation.

Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene.

Deletion mutations ending in the 5'-flanking sequences of the Saccharomyces cerevisiae SUP4-o gene have been analyzed for their effects on gene expression. This ochre-suppressing tRNATyr gene was cloned into a S. cerevisiae centromeric plasmid, and its level of in vivo expression was monitored by observing the suppressor phenotype of the gene after transformation into S. cerevisiae. A deletion mutant that retains only four base pairs of the 5'-flanking sequence is profoundly deficient in expression; deletion mutants extending to positions -18, -17, -16, or -15 are moderately deficient; deletion mutants extending to positions -36 or -27 are slightly defective; and mutants retaining more than 60 base pairs of the original 5'-flanking DNA are expressed normally. In some cases, the cloning procedure led to the introduction of multiple BamHI linkers at the SUP4-o-vector fusion site, and in one instance, the resulting structure dramatically affects gene function: the presence of three linkers abutting a -18 deletion completely inhibits the in vivo expression of SUP4-o. In contrast, three linkers that abut a -77 deletion have no effect on in vivo expression. The template properties of these plasmids in a homologous in vitro transcription system parallel the levels of in vivo expression, suggesting that the mutations predominantly affect transcription. The data demonstrate that there are significant functional constraints on the 5'-flanking sequences of this RNA polymerase III-transcribed gene. The dramatic effects of the multiple linker insertion at position -18 suggest that there may be extensive melting of the DNA in this region during normal transcription initiation.

Nucleotide sequence of a 1.1 kb fragment of the pea chloroplast genome containing three tRNA genes, one of which is located within an open reading frame of 91 codons.

The nucleotide sequence of a 1082 bp fragment from the pea (Pisum sativum) chloroplast genome is presented. This fragment contains genes for tRNAGlu, tRNATyr and tRNAAsp as well as an open reading frame (ORF) of 91 codons on one strand and two ORFs of 52 and 59 codons on the complementary strand. The tRNAAsp gene is located entirely within the ORF of 91 codons. The first 366 bp of the fragment correspond to 376 bp at one end of a recently published (1) sequence from the broad bean (Vicia faba) chloroplast genome. These regions contain the tRNAGlu and tRNATyr genes, which are identical and separated by 60 bp in both species. These two genes are probably cotranscribed. The intergenic regions in the corresponding segments from the two species are, except for a 10 bp deletion in the pea sequence, 94% homologous.

Nucleotide sequence of a 2 kbp BamH I fragment of Vicia faba chloroplast DNA containing the genes for threonine, glutamic acid and tyrosine transfer RNAs.

The entire nucleotide sequence of a 2014 bp BamH I fragment from broad bean (Vicia faba) chloroplast DNA containing the genes for tRNAThr (trnT), tRNAGlu (trnE) and tRNATyr (trnY) has been determined. The tRNAGlu and tRNATyr genes are separated by only 60 bp and are probably part of the same transcriptional unit. The tRNAThr gene is located on the complementary strand, 876 bp away from the tRNAGlu gene. This fragment also contains an open reading frame of 82 codons, as well as a series of AT-rich, direct and inverted repeats.