Identification of a mutation in the mitochondrial tRNA(Cys) gene associated with mitochondrial encephalopathy.
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Base Sequence↗
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The complete nucleotide sequence of the mitochondrial genome of sugar beet (Beta vulgaris L.) reveals a novel gene for tRNA(Cys)(GCA).
We determined the complete nucleotide sequence of the mitochondrial genome of an angiosperm, sugar beet (Beta vulgaris cv TK81-O). The 368 799 bp genome contains 29 protein, five rRNA and 25 tRNA genes, most of which are also shared by the mitochondrial genome of Arabidopsis thaliana, the only other completely sequenced angiosperm mitochondrial genome. However, four genes identified here (namely rps13, trnF-GAA, ccb577 and trnC2-GCA) are missing in Arabidopsis mitochondria. In addition, four genes found in Arabidopsis (ccb228, rpl2, rpl16 and trnY2-GUA) are entirely absent in sugar beet or present only in severely truncated form. Introns, duplicated sequences, additional reading frames and inserted foreign sequences (chloroplast, nuclear and plasmid DNA sequences) contribute significantly to the overall size of the sugar beet mitochondrial genome. Nevertheless, 55.6% of the genome has no obvious features of information. We identified a novel tRNA(Cys) gene (trnC2-GCA) which shows no sequence homology with any tRNA(Cys) genes reported so far in higher plants. Intriguingly, this tRNA gene is actually transcribed into a mature tRNA, whereas the native tRNA(Cys) gene (trnC1-GCA) is most likely a pseudogene.
Arabidopsis↗
The evolutionary history of Cys-tRNACys formation.
The recent discovery of an alternate pathway for indirectly charging tRNA(Cys) has stimulated a re-examination of the evolutionary history of Cys-tRNA(Cys) formation. In the first step of the pathway, O-phosphoseryl-tRNA synthetase charges tRNA(Cys) with O-phosphoserine (Sep), a precursor of the cognate amino acid. In the following step, Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep to Cys in a tRNA-dependent reaction. The existence of such a pathway raises several evolutionary questions, including whether the indirect pathway is a recent evolutionary invention, as might be implied from its localization to the Euryarchaea, or, as evidence presented here indicates, whether this pathway is more ancient, perhaps already in existence at the time of the last universal common ancestral state. A comparative phylogenetic approach is used, combining evolutionary information from protein sequences and structures, that takes both the signature of horizontal gene transfer and the recurrence of the full canonical phylogenetic pattern into account, to document the complete evolutionary history of cysteine coding and understand the nature of this process in the last universal common ancestral state. Resulting from the historical study of tRNA(Cys) aminoacylation and the integrative perspective of sequence, structure, and function are 3D models of O-phosphoseryl-tRNA synthetase and SepCysS, which provide experimentally testable predictions regarding the identity and function of key active-site residues in these proteins. The model of SepCysS is used to suggest a sulfhydrylation reaction mechanism, which is predicted to occur at the interface of a SepCysS dimer.
Amino Acid Sequence↗
Lack of a functional plastid tRNA(Cys) gene is associated with loss of photosynthesis in a lineage of parasitic plants.
We recently reported that the gene for chloroplast tRNA(Cys)(GCA) is a pseudogene in the plastid DNA of Epifagus virginiana, a non-photosynthetic parasitic flowering plant in the family Orobanchaceae. Since this is the only tRNA(Cys) gene in the plastid genome, and since Epifagus appears to possess a functional plastid translational apparatus, it seems probable that nuclear-encoded tRNAs are imported into plastids to effect translation. In this study we have surveyed species closely related to Epifagus to establish how widespread the loss of this tRNA gene has been. We find that Conopholis americana, another non-photosynthetic parasite, lacks the gene altogether, but that seven closely-related photosynthetic plants (both parasitic and free-living) maintain an intact chloroplast tRNA(Cys) gene. Thus, the tRNA(Cys) gene appears to have become non-functional at the same time that photosynthetic ability was lost. This may be because the levels of putatively imported tRNAs are sufficient to meet the demands of plastid gene expression under nonphotosynthetic conditions only.
Animals↗
Aminoacylation of an unusual tRNA(Cys) from an extreme halophile.
The extreme halophile Halobacterium species NRC-1 overcomes external near-saturating salt concentrations by accumulating intracellular salts comparable to those of the medium. This raises the fundamental question of how halophiles can maintain the specificity of protein-nucleic acid interactions that are particularly sensitive to high salts in mesophiles. Here we address the specificity of the essential aminoacylation reaction of the halophile, by focusing on molecular recognition of tRNA(Cys) by the cognate cysteinyl-tRNA synthetase. Despite the high salt environments of the aminoacylation reaction, and despite an unusual structure of the tRNA with an exceptionally large dihydrouridine loop, we show that aminoacylation of the tRNA proceeds with a catalytic efficiency similar to that of its mesophilic counterparts. This is manifested by an essentially identical K(m) for tRNA to those of the mesophiles, and by recognition of the same nucleotide determinants that are conserved in evolution. Interestingly, aminoacylation of the halophile tRNA(Cys) is more closely related to that of bacteria than eukarya by placing a strong emphasis on features of the tRNA tertiary core. This suggests an adaptation to the highly negatively charged tRNA sugar-phosphate backbone groups that are the key elements of the tertiary core.
Acylation↗
Mutations in the yeast mitochondrial RNA polymerase specificity factor, Mtf1, verify an essential role in promoter utilization.
The yeast mitochondrial RNA polymerase (RNAP) is a two-subunit enzyme composed of a catalytic core (Rpo41) and a specificity factor (Mtf1) encoded by nuclear genes. Neither subunit on its own interacts with promoter DNA, but the combined holo-RNAP recognizes and selectively initiates from promoters related to the consensus sequence ATATAAGTA. To pursue the question of why Rpo41, which resembles the single polypeptide RNAPs from bacteriophage T7 and T3, requires a separate specificity factor, we analyzed a collection of Mtf1 point mutations that confer an in vivo petite phenotype. These mutant proteins are able to interact with Rpo41 and are capable of nearly wild type levels of initiation in vitro with a consensus promoter-containing template (14 S rRNA). However, the petite phenotype of two mutants can be explained by the fact that they exhibit dramatic transcriptional defects on non-consensus promoters. Y54F is incapable of transcribing the weak tRNA(Cys) promoter, and C192F cannot transcribe either tRNA(Cys) or the variant COX2 promoter from linear DNA templates. Transcription of the tRNA(Cys) promoter by both mutants was significantly corrected by addition of an initiating dinucleotide primer or by supercoiling the DNA template. These results establish the critical role of Mtf1 in promoter recognition and initiation of transcription.
Amino Acid Substitution↗
Cysteine tRNAs of plant origin as novel UGA suppressors.
We have isolated and sequenced chloroplast (chl) and cytoplasmic (cyt) cysteine tRNAs from Nicotiana rustica. Both tRNAs carry a GCA anticodon but beyond that differ considerably in their nucleotide sequences. One obvious distinction resides in the presence of N6-isopentenyladenosine (i6A) and 1-methylguanosine (m1G) at position 37 in chl and cyt tRNA(Cys) respectively. In order to study the potential suppressor activity of tRNAs(Cys) we used in vitro synthesized zein mRNA transcripts in which an internal UGA stop codon had been placed in either the tobacco rattle virus (TRV)- or tobacco mosaic virus (TMV)-specific codon context. In vitro translation was carried out in a messenger- and tRNA-dependent wheat germ extract. Both tRNA(Cys) isoacceptors stimulate read-through over the UGA stop codon, however, chl tRNA(GCA)Cys is more efficient than the cytoplasmic counterpart. The UGA in the two viral codon contexts is suppressed to about the same extent by either of the two tRNAs(Cys), whereas UGA in the beta-globin context is not recognized at all. The interaction of tRNA(GCA)Cys with UGA requires an unconventional G:A base pair in the wobble position, as postulated earlier for plant tRNA(G psi A)Tyr misreading the UAA stop codon. This is the first case that a cysteine-accepting tRNA has been characterized as a natural UGA suppressor.
Amino Acid Sequence↗
Reassigning cysteine in the genetic code of Escherichia coli.
We investigated directed deviations from the universal genetic code. Mutant tRNAs that incorporate cysteine at positions corresponding to the isoleucine AUU, AUC, and AUA and methionine AUG codons were introduced in Escherichia coli K12. Missense mutations at the cysteine catalytic site of thymidylate synthase were systematically crossed with synthetic suppressor tRNACys genes coexpressed from compatible plasmids. Strains harboring complementary codon/anticodon associations could be stably propagated as thymidine prototrophs. A plasmid-encoded tRNACys reading the codon AUA persisted for more than 500 generations in a strain requiring its suppressor activity for thymidylate biosynthesis, but was eliminated from a strain not requiring it. Cysteine miscoding at the codon AUA was also enforced in the active site of amidase, an enzyme found in Helicobacter pylori and not present in wild-type E. coli. Propagating the amidase missense mutation in E. coli with an aliphatic amide as nitrogen source required the overproduction of Cys-tRNA synthetase together with the complementary suppressor tRNACys. The toxicity of cysteine miscoding was low in all our strains. The small size and amphiphilic character of this amino acid may render it acceptable as a replacement at most protein positions and thus apt to overcome the steric and polar constraints that limit evolution of the genetic code.
Amidohydrolases↗
The hypotrichous ciliate Euplotes octocarinatus has only one type of tRNACys with GCA anticodon encoded on a single macronuclear DNA molecule.
Deviations from the universal genetic code have evolved independently several times in ciliated protozoa. Thus, in some species UAA and UAG are no longer used as termination codons, but are read as glutamine, whereas in the genus Euplotes , UGA is translated as cysteine. We have investigated the nature of the tRNACys isoacceptor responsible for decoding UGA in Euplotes cells. Southern hybridization analyses indicated that a single DNA molecule of 630 bp encoding tRNACys exists in the macronucleus of Euplotes octocarinatus . Cloning and sequencing of this fragment revealed that it contains only one copy of a tRNACys gene, which codes for a normal tRNACys with GCA anticodon. This is the first report of the characterization of a tRNA gene in any hypotrichous ciliate. It contains putative signals for initiation and termination of transcription by RNA polymerase III and can be transcribed efficiently in vitro in HeLa cell nuclear extract. Intensive studies on the DNA and tRNA level involving PCR analyses have not disclosed the existence of any tRNA Cys isoacceptor with UCA or ICA anticodons. Translation of the UGA codon by tRNA sub GCA sup Cys necessitates a G:A mispairing in the first anticodon position. We discuss a number of aspects which might contribute to the finding that a near-cognate tRNA isoacceptor efficiently translates the UGA stop codon.
Animals↗
Structures of two bacterial prolyl-tRNA synthetases with and without a cis-editing domain.
Prolyl-tRNA synthetases (ProRSs) are unique among synthetases in that they have diverse architectures, notably the variable presence of a cis-editing domain homologous to the freestanding deacylase proteins YbaK and ProX. Here, we describe crystal structures of two bacterial ProRSs from the pathogen Enterococcus faecalis, which possesses an editing domain, and from Rhodopseudomonas palustris, which does not. We compare the overall structure and binding mode of ATP and prolyl-adenylate with those of the archael/eukaryote-type ProRS from Thermus thermophilus. Although structurally more homologous to YbaK, which preferentially hydrolyzes Cys-tRNA(Pro), the editing domain of E. faecalis ProRS possesses key elements similar to ProX, with which it shares the activity of hydrolyzing Ala-tRNA(Pro). The structures give insight into the complex evolution of ProRSs, the mechanism of editing, and structural differences between prokaryotic- and eukaryotic-type ProRSs that can be exploited for antibiotic design.
Adenosine Triphosphate↗
Use of biotinylated-cysteinyl-tRNA as a non-RI probe in protein synthesis.
A convenient method for the preparation of biotinylated aminoacyl-tRNA to use in the non-radioisotopic (non-RI) detection of cell-free translation products was developed. After aminoacylation of E. coli tRNA(Cys) with L-cystein, its sulfhydryl group was modified with N-(6-[Biotinamide]hexyl)-3'-(2'-pyridyl dithio) propionamide or 1-Biotin amido-4-(4'-[maleimidomethyl] cyclohexane-carboxamido) butane. These biotin-labelled cysteinyl-tRNA are expected to function as the non-RI probe for protein synthesis equally to or even better than the biotinylated lysyl-tRNA which is now commercially available.
Biotin↗
[Structure analysis of the mitochondrial tRNA(Cys) gene and the original region of light-strand replication from carp].
In this study, we have determined the nucleotide sequences of the cysteine tRNA gene and the original region of light-strand replication from carp (Cyprinus carpio L.) mitochondrion, which were further represented respectively in cloverleaf secondary structure and stem loop structure. By comparing the nucleotide sequences of 5 vertebrate tRNA(Cys) genes, it is found that carp mitochondrial tRNA(Cys) gene have many unusual structural features differing from those of cytoplasmic tRNA(Cys) gene. The carp mitochondrial original region of light-strand replication consists of 36 bases which include 11 base pairs in its stem and 14 bases in its loop. By making a comparison between the nucleotide sequences of the carp original region of light-strand replication and those of other 10 vertebrates, it is further found that stem sequence of carp is very conservative while loop sequence is very variable. This result indicates that stem structure may play an important role in the replication of light strand.
Animals↗
Mitochondrial tRNA(Cys) gene mutation (A5814G): a second family with mitochondrial encephalopathy.
We report an Italian family with maternally inherited encephalomyopathy including progressive external ophthalmoplegia, seizures, and neurophysiological evidence of brainstem dysfunction. Mitochondrial DNA analysis showed a heteroplasmic point mutation at position 5814 in the tRNA gene for cysteine (A5814G), previously reported in a 5-year-old girl of Portuguese origin. The mutation was very abundant (> 95%) in both muscle and blood from the proposita and was present in lower proportions (average 85 +/- 6%) in blood from three less severely affected maternal relatives. This observation confirms pathogenicity for the A5814G mutation.
Adult↗
Probing a tRNA core that contributes to aminoacylation.
The contribution of the tRNA "core" to aminoacylation is beginning to be recognized. One example is the core region of Escherichia coli tRNA(Cys), which has been shown by biochemical studies to be important for aminoacylation. This core has several layers of unusual base-pairs, which are revealed by the recent crystal structure of the tRNA complexed with the elongation factor EF-Tu and an analog of GTP. One of these layers consists of a 9:[13:22] base-triple, rather than the 46:[13:22] or 45:[13:22] base-triple that is commonly observed in tRNA structure. Because 13:22 is an important element in aminoacylation of E. coli tRNA(Cys), a better understanding of its structure in the tRNA core will shed light on its role in aminoacylation. In this study, we used the phage T7 transcript of the tRNA as a substrate. We probed the structure of 13:22 by dimethyl sulfate and tested its partner in a base-triple by generating mutations that could be assayed for aminoacylation. The results of this study in general are in a better agreement with a 46:[13:22] base-triple that we previously proposed. Although these results are not interpreted as direct proof for the 46:[13:22] base-triple, they shed new light on features of the tRNA core that are important for aminoacylation.
Base Pairing↗
Recognition of tRNA(Cys) by Escherichia coli cysteinyl-tRNA synthetase.
A study of the recognition of tRNA(Cys) by Escherichia coli cysteinyl-tRNA synthetase using in vivo and in vitro methods was performed. All three anticodon nucleotides, the discriminator nucleotide (73), and some elements within the tertiary domain (the D stem/loop, the T psi C stem/loop, and the variable loop) are important for recognition; the anticodon stem and acceptor stem appear to contain no essential elements. A T7 RNA polymerase transcript corresponding to tRNA(Cys) is only a 5.5-fold worse substrate than native tRNA(Cys) (in terms of the specificity constant, kcat/Km), mainly due to an increase in the value of Km for the transcript. The greatest loss of specificity caused by mutation of a single nucleotide occurs when the discriminator U73 is changed; kcat/Km declines 3-4 orders of magnitude depending on the substitution. Mutations in the wobble nucleotide of the anticodon also cause reductions in the specificity constant of 3 orders of magnitude, while mutations in the other anticodon nucleotides caused lesser effects. Interestingly, a C35A mutation (with the phenylalanine anticodon GAA) had no effect on aminoacylation by the cysteinyl-tRNA synthetase. Several amber suppressor tRNAs were constructed whose in vivo identity did not correlate with their in vitro specificity, indicating the need for both types of experiments to understand the factors which maintain tRNA specificity.
Acylation↗
Mitochondrial tRNA(Cys) mutation A5823G in a patient with motor neuron disease and temporal lobe epilepsy.
We discovered a new homoplasmic mutation in the mitochondrial cysteine tRNA of a 60-year-old Caucasian male suffering from asymmetrical pure lower motor neuron disease (MND) and temporal lobe epilepsy (TLE). Furthermore, titrations with Amytal, an inhibitor of NADH:CoQ oxidoreductase, revealed mild mitochondrial dysfunction in skeletal muscle tissue, which was described in patients with MND in an earlier report. The mutation was undetectable in 155 Caucasian controls of both sexes, in 40 MND patients and in 13 individuals suffering from TLE. It was, however, detected in a heteroplasmic state in the patient's mother, who did not suffer from a neurological disorder. Since this rare mutation affected a nonconserved base position and was not observed in MND or TLE materials, its relation to disease remains unclear.
Blotting, Southern↗
Conservation of a tRNA core for aminoacylation.
The core region of Escherichia coli tRNA(Cys)is important for aminoacylation of the tRNA. This core contains an unusual G15:G48 base pair, and three adenosine nucleotides A13, A22 and A46 that are likely to form a 46:[13:22] adenosine base triple. We recently observed that the 15:48 base pair and the proposed 46:[13:22] triple are structurally and functionally coupled to contribute to aminoacylation. Inspection of a database of tRNA sequences shows that these elements are only found in one other tRNA, the Haemophilus influenzae tRNA(Cys). Because of the complexity of the core, conservation of sequence does not mean conservation of function. We here tested whether the conserved elements in H. influenzae tRNA(Cys)were also important for aminoacylation of H. influenzae tRNA(Cys). We cloned and purified a recombinant H. influenzae cysteine-tRNA synthe-tase and showed that it depends on 15:48 and 13, 22 and 46 in a relationship analogous to that of E. coli cysteine-tRNA synthetase. The functional conservation of the tRNA core is correlated with sequence conservation between E.coli and H.influenzae cysteine-tRNA synthetases. As the genome of H. influenzae is one of the smallest and may approximate a small autonomous entity in the development of life, the dependence of this genome on G15:G48 and its coupling with the proposed A46:[A13:A22] triple for aminoacylation with cysteine suggests an early role of these motifs in the evolution of decoding genetic information.
Acylation↗
Vicariant patterns of fragmentation among gekkonid lizards of the genus Teratoscincus produced by the Indian collision: A molecular phylogenetic perspective and an area cladogram for Central Asia.
A well-supported phylogenetic hypothesis is presented for gekkonid lizards of the genus Teratoscincus. Phylogenetic relationships of four of the five species are investigated using 1733 aligned bases of mitochondrial DNA sequence from the genes encoding ND1 (subunit one of NADH dehydrogenase), tRNA(Ile), tRNA(Gln), tRNA(Met), ND2, tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), tRNA(Tyr), and COI (subunit I of cytochrome c oxidase). A single most parsimonious tree depicts T. przewalskii and T. roborowskii as a monophyletic group, with T. scincus as their sister taxon and T. microlepis as the sister taxon to the clade containing the first three species. The aligned sequences contain 341 phylogenetically informative characters. Each node is supported by a bootstrap value of 100% and the shortest suboptimal tree requires 29 additional steps. Allozymic variation is presented for proteins encoded by 19 loci but these data are largely uninformative phylogenetically. Teratoscincus species occur on tectonic plates of Gondwanan origin that were compressed by the impinging Indian Subcontinent, resulting in massive montane uplifting along plate boundaries. Taxa occurring in China (Tarim Block) form a monophyletic group showing vicariant separation from taxa in former Soviet Central Asia and northern Afghanistan (Farah Block); alternative biogeographic hypotheses are statistically rejected. This vicariant event involved the rise of the Tien Shan-Pamir and is well dated to 10 million years before present. Using this date for separation of taxa occurring on opposite sides of the Tien Shan-Pamir, an evolutionary rate of 0.57% divergence per lineage per million years is calculated. This rate is similar to estimates derived from fish, bufonid frogs, and agamid lizards for the same region of the mitochondrial genome ( approximately 0.65% divergence per lineage per million years). Evolutionary divergence of the mitochondrial genome has a surprisingly stable rate across vertebrates.
Animals↗