tRNAs are imported into mitochondria of Trypanosoma brucei independently of their genomic context and genetic origin.

The mitochondrial genome of Trypanosoma brucei does not encode any identifiable tRNAs. Instead, mitochondrial tRNAs are synthesized in the nucleus and subsequently imported into mitochondria. In order to analyse the signals which target the tRNAs into the mitochondria, an in vivo import system has been developed: tRNA variants were expressed episomally and their import into mitochondria assessed by purification and nuclease treatment of the mitochondrial fraction. Three tRNA genes were tested in this system: (i) a mutated version of the trypanosomal tRNA(Tyr); (ii) a cytosolic tRNA(His) of yeast; and (iii) a human cytosolic tRNA(Lys). The tRNAs were expressed in their own genomic context, or containing various lengths of the 5'-flanking sequence of the trypanosomal tRNA(Tyr) gene. In all cases efficient import of each of the tRNAs was observed. We independently confirmed the mitochondrial import of the yeast tRNA(His), since in organello [alpha-32P]ATP-labelling of the 3'-end of the tRNA was inhibited by carboxyatractyloside, a highly specific inhibitor of the mitochondrial adenine nucleotide translocator. Import of heterologous tRNAs in their own genomic contexts supports the conclusion that no specific targeting signals are necessary to import tRNAs into mitochondria of T. brucei, but rather that the tRNA structure itself is sufficient to specify import.     

Bovine mitochondrial tRNAPhe, tRNASer (AGY) and tRNASer (UCN): preparation using a new detection method and their properties in aminoacylation

Bovine mitochondrial tRNAPhe, tRNASer (AGY), and tRNASer (UCN) possessing unusual structures were purified using a new hybridization assay system and their properties in aminoacylation were examined. Bovine mitochondrial phenyl-alanyl- and seryl-tRNA synthetases could aminoacylate the same amino acid-specific tRNAs obtained not only from the mitochondria but also from other sources such as E. coli, Thermus thermophilus, bovine and yeast cytosols and archaebacteria, Sulfolobus acidocaldarius. On the contrary, none of both bacterial and cytosolic synthetases could aminoacylate the same amino acid specific tRNAs from the heterologous sources with some exceptions. We consider that the bovine mitochondrial aminoacyl-tRNA synthetases have considerably simple recognition mechanism toward the substrate tRNAs compared with the non-mitochondrial ones. This mechanism may be correlated with the occurrence of structural varieties of the mitochondrial tRNA species with unusual structures.     

Cytosolic yeast tRNA(His) is covalently modified when imported into mitochondria of Trypanosoma brucei.

The mitochondrial genome of Trypanosoma brucei does not encode any tRNAs. Instead, mitochondrial tRNAs are synthesized in the nucleus and subsequently imported into mitochondria. The great majority of mitochondrial tRNAs have cytosolic counterparts showing identical primary sequences. The only difference found between mitochondrial and cytosolic isotypes of the tRNAs are mitochondria-specific nucleotide modifications which appear to be a common feature of imported tRNAs in trypanosomes. In this study, a mutated yeast cytosolic tRNAHis was expressed in trypanosomes and its import phenotype was analyzed by cell fractionation and nuclease treatment of intact mitochondria. Furthermore, cytosolic and mitochondrial isotypes of the yeast tRNA(His) were specifically labeled and analyzed by limited alkaline hydrolysis. These experiments revealed the presence of mitochondria-specific nucleotide modifications in the yeast tRNA(His). The positions of the modifications were determined by direct enzymatic sequencing of the tRNA(His) and shown to correspond to the ultimate and penultimate nucleotides before the anticodon, the same relative positions which are modified in the mitochondrial isotype of trypanosomal tRNA(Tyr). The results demonstrate that covalent modification of tRNAs; in trypanosomal mitochondria can be used, in analogy to processing of precursor proteins during mitochondrial protein import, as a marker for import of both endogenous and heterologous tRNAs.     

Characterization of a yeast mitochondrial locus necessary for tRNA biosynthesis. Deletion mapping and restriction mapping studies

Yeast mitochondrial DNA codes for a complete set of tRNAs. Although most components necessary for the biosynthesis of mitochondrial tRNA are coded by nuclear genes, there is one genetic locus on mitochondrial DNA necessary for the synthesis of mitochondrial tRNAs other than the mitochondrial tRNA genes themselves. Characterization of mutants by deletion mapping and restriction enzyme mapping studies has provided a precise location of this yeast mitochondrial tRNA synthesis locus. Deletion mutants retaining various segments of mitochondrial DNA were examined for their ability to synthesize tRNAs from the genes they retain. A subset of these strains was also tested for the ability to provide the tRNA synthesis function in complementation tests with deletion mutants unable to synthesize mature mitochondrial tRNAs. By correlating the tRNA synthetic ability with the presence or absence of certain wild-type restriction fragments, we have confined the locus to within 780 base pairs of DNA located between the tRNAMetf gene and tRNAPro gene, at 29 units on the wild-type map. Heretofore, no genetic function or gene product had been localized in this area of the yeast mitochondrial genome.     

Identification of nuclear encoded precursor tRNAs within the mitochondrion of Trypanosoma brucei.

RNAs that function in mitochondria are typically encoded by the mitochondrial DNA. However, the mitochondrial tRNAs of Trypanosoma brucei are encoded by the nuclear DNA and therefore must be imported into the mitochondrion. It is becoming evident that RNA import into mitochondria is phylogenetically widespread and is essential for cellular processes, but virtually nothing is known about the mechanism of RNA import. We have identified and characterized mitochondrial precursor tRNAs in T. brucei. The identification of mitochondrially located precursor tRNAs clearly indicates that mitochondrial tRNAs are imported as precursors. The mitochondrial precursor tRNAs hybridize to cloned nuclear tRNA genes, label with [alpha-32P]CTP using yeast tRNA nucleotidyltransferase and in isolated mitochondria via an endogenous nucleotidyltransferase-like activity, and are processed to mature tRNAs by Escherichia coli and yeast mitochondrial RNase P. We show that T. brucei mitochondrial extract contains an RNase P activity capable of processing a prokaryotic tRNA precursor as well as the T. brucei tRNA precursors. Precursors for tRNA(Asn) and tRNA(Leu) were detected on Northern blots of mitochondrial RNA, and the 5' ends of these RNAs were characterized by primer extension analysis. The structure of the precursor tRNAs and the significance of nuclear encoded precursor tRNAs within the mitochondrion are discussed.     

Yeast mitochondrial methionine initiator tRNA: characterization and nucleotide sequence.

Two methionine tRNAs from yeast mitochondria have been purified. The mitochondrial initiator tRNA has been identified by formylation using a mitochondrial enzyme extract. E. coli transformylase however, does not formylate the yeast mitochondrial initiator tRNA. The sequence was determined using both 32P-in vivo labeled and 32P-end labeled mt tRNAf(Met). This tRNA, unlike N. crassa mitochondrial tRNAf(Met), has two structural features typical of procaryotic initiator tRNAs: (i) it lacks a Watson-Crick base-pair at the end of the acceptor stem and (ii) has a T-psi-C-A sequence in loop IV. However, both yeast and N. crassa mitochondrial initiator tRNAs have a U11:A24 base-pair in the D-stem unlike procaryotic initiator tRNAs which have A11:U24. Interestingly, both mitochondrial initiator tRNAs, as well as bean chloroplast tRNAf(Met), have only two G:C pairs next to the anticodon loop, unlike any other initiator tRNA whatever its origin. In terms of overall sequence homology, yeast mitochondrial tRNA(Met)f differs from both procaryotic or eucaryotic initiator tRNAs, showing the highest homology with N. crassa mitochondrial initiator 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.     

Study of yeast mitochondrial tRNAs by two-dimensional polyacrylamide gel electrophoresis: characterization of isoaccepting species and search for imported cytoplasmic tRNAs.

By two-dimensional polyacrylamide gel electrophoresis, yeast mitochondrial tRNA is fractionated into 27 major species. All but 6 of them migrate distinctly from cytoplasmic tRNAs. Migration of mitochondrial DNA-coded mitochondrial tRNAs shows the occurence of only one cytoplasmic tRNA in mitochondria. Several mitochondrial tRNA spots are identified on the electrophoregrams, some of them show isoaccepting species (Val, Ser, Met, Leu). It is suggested that there are sufficient mitochondrial tRNA genes on yeast mitochondrial DNA to allow mitochondrial protein biosynthesis by the mitochondrial tRNAs alone. Guanosine + Cytidine content and rate base composition are reported for some individual species. Mitochondrial tRNAPhe lacks Ribothymidine.     

Transfer RNA genes in the cap-oxil region of yeast mitochondrial DNA.

A cytoplasmic "petite" (rho-) clone of Saccharomyces cerevisiae has been isolated and found through DNA sequencing to contain the genes for cysteine, histidine, leucine, glutamine, lysine, arginine, and glycine tRNAs. This clone, designated DS502, has a tandemly repeated 3.5 kb segment of the wild type genome from 0.7 to 5.6 units. All the tRNA genes are transcribed from the same strand of DNA in the direction cap to oxil. The mitochondrial DNA segment of DS502 fills a sequence gap that existed between the histidine and lysine tRNAs. The new sequence data has made it possible to assign accurate map positions to all the tRNA genes in the cap-oxil span of the yeast mitochondrial genome. A detailed restriction map of the region from 0 to 17 map units along with the locations of 16 tRNA genes have been determined. The secondary structures of the leucine and glutamine tRNAs have been deduced from their gene sequences. The leucine tRNA exhibits 64% sequence homology to an E. coli leucine tRNA.     

Transfer RNA genes in mitochondrial DNA of grande (wild-type) yeast

We have used transfer RNA-DNA hybridization to show that seven tRNAs, i.e. tyrosyl, glutamyl, aspartyl, prolyl, lysyl, histidyl and seryl, hybridize with grande yeast mitochondrial DNA. These tRNA species are in addition to the seven which we previously showed to be gene products of mitochondrial DNA. Escherichia coli aminoacyl-synthetase preparations also were shown to catalyze specific acylation of yeast mitochondrial leucyl and tyrosyl-tRNA, but not of the isoaccepting tRNAs localized in the cell supernatant. Cytoplasmic tRNAs were found to be present in our purified mitochondrial preparations.     

Primary and spatial structure of tRNA

The recent achievements in studying of structure of tRNA are considered in the present paper. A brief analysis of the new methods for sequencing tRNA was carried out. Due to the development of these methods about 300 tRNA primary structures have been determined. Comparison of the primary tRNA structures gives us the possibility to divide them into seven classes: prokaryotic initiator tRNAs and eukaryotic initiator tRNAs; prokaryotic elongator tRNAs and eukaryotic elongator tRNAs; archaebacterial tRNAs; and mitochondrial tRNAs of lower and higher eukaryotes. Structural properties of the tRNAs of each of these classes are discussed. The second part of the paper is devoted to the three-dimensional structure of tRNA. Recent data in this field obtained by X-ray crystallographic technique as well as by high-resolution NMR and chemical modification methods are reviewed.     

Aminoacylation and conformational properties of yeast mitochondrial tRNA mutants with respiratory deficiency

We report the identification and characterization of eight yeast mitochondrial tRNA mutants, located in mitochondrial tRNA(Gln), tRNA(Arg2), tRNA(Ile), tRNA(His), and tRNA(Cys), the respiratory phenotypes of which exhibit various degrees of deficiency. The mutations consist in single-base substitutions, insertions, or deletions, and are distributed all over the tRNA sequence and structure. To identify the features responsible for the defective phenotypes, we analyzed the effect of the different mutations on the electrophoretic mobility and efficiency of acylation of the mutated tRNAs in comparison with the respective wild-type molecules. Five of the studied mutations determine both conformational changes and defective acylation, while two have neither or limited effect. However, variations in structure and acylation are not necessarily correlated; the remaining mutation affects the tRNA conformation, but not its acylation properties. Analysis of tRNA structures and of mitochondrial and cytoplasmic yeast tRNA sequences allowed us to propose explanations for the observed defects, which can be ascribed to either the loss of identity nucleotides or, more often, of specific secondary and/or tertiary interactions that are largely conserved in native mitochondrial and cytoplasmic tRNAs.     

A model for the tertiary structure of mammalian mitochondrial transfer RNAs lacking the entire 'dihydrouridine' loop and stem

The mammalian mitochondrial tRNA(AGY)Ser is unique in lacking the entire dihydrouridine arm. This reduces its secondary structure to a 'truncated cloverleaf'. Experimental evidence on the tertiary structure has been obtained by chemically probing the conformation of both the bovine and human species in their native conformation and at various stages of denaturation. A structural model of the bovine tRNA is presented based on the results of this chemical probing, on a comparison between nine homologous 'truncated cloverleaf' secondary structures and on analogies with the crystal structure of yeast phenylalanine tRNA. The proposed structure is very similar in shape to that of yeast tRNA(Phe) but is slightly smaller in size. It is defined by a unique set of tertiary interactions. Structural considerations suggest that other mammalian mitochondrial tRNAs have smaller dimensions as well.     

Yeast mitochondrial and cytoplasmic valyl-tRNA synthetases

Yeast mitochondrial tRNA synthetase has been partially purified and chromatographic, catalytic and antigenic properties have been compared to the cytoplasmic homologous enzyme from yeast. No significant differences could be observed between the two enzymes with respect to their behaviour during ammonium sulfate precipitation or in chromatographic separation on DEAE cellulose, hydroxylapatite and Sephadex G 200. The Km of the two enzymes toward tRNAs from yeast mitochondria, yeast cytoplasm or E. coli are pratically identical. The antigenic properties of the two enzymes are very similar; antisera against either the mitochondria or the cytoplasmic enzyme lead to the inhibition of their catalytic properties. The mitochondrial ValRS is formed by a single polypeptide chain whose molecular weight is 125,000 daltons, a value very close to that of the yeast cytoplasmic enzyme.     

Improved systematic tRNA gene annotation allows new insights into the evolution of mitochondrial tRNA structures and into the mechanisms of mitochondrial genome rearrangements

Transfer RNAs (tRNAs) are present in all types of cells as well as in organelles. tRNAs of animal mitochondria show a low level of primary sequence conservation and exhibit 'bizarre' secondary structures, lacking complete domains of the common cloverleaf. Such sequences are hard to detect and hence frequently missed in computational analyses and mitochondrial genome annotation. Here, we introduce an automatic annotation procedure for mitochondrial tRNA genes in Metazoa based on sequence and structural information in manually curated covariance models. The method, applied to re-annotate 1876 available metazoan mitochondrial RefSeq genomes, allows to distinguish between remaining functional genes and degrading 'pseudogenes', even at early stages of divergence. The subsequent analysis of a comprehensive set of mitochondrial tRNA genes gives new insights into the evolution of structures of mitochondrial tRNA sequences as well as into the mechanisms of genome rearrangements. We find frequent losses of tRNA genes concentrated in basal Metazoa, frequent independent losses of individual parts of tRNA genes, particularly in Arthropoda, and wide-spread conserved overlaps of tRNAs in opposite reading direction. Direct evidence for several recent Tandem Duplication-Random Loss events is gained, demonstrating that this mechanism has an impact on the appearance of new mitochondrial gene orders.     

Pathogenic mutations in antisense mitochondrial tRNAs

Pathogenic mutations in mitochondrial tRNAs are 6.5 times more frequent than in other mitochondrial genes. This suggests that tRNA mutations perturb more than one function. A potential additional tRNA gene function is that of templating for antisense tRNAs. Pathogenic mutations weaken cloverleaf secondary structures of sense tRNAs. Analyses here show similar effects for most antisense tRNAs, especially after adjusting for associations between sense and antisense cloverleaf stabilities. These results imply translational activity by antisense tRNAs. For sense tRNAs Ala and Ser UCN, pathogenicity associates as much with sense as with antisense cloverleaf formation. For tRNA Pro, pathogenicity seems associated only with antisense, not sense tRNA cloverleaf formation. Translational activity by antisense tRNAs is expected for the 11 antisense tRNAs processed by regular sense RNA maturation, those recognized by their cognate amino acid’s tRNA synthetase, and those forming relatively stable cloverleaves as compared to their sense counterpart. Most antisense tRNAs probably function routinely in translation and extend the tRNA pool (extension hypothesis); others do not (avoidance hypothesis). The greater the expected translational activity of an antisense tRNA, the more pathogenic mutations weaken its cloverleaf secondary structure. Some evidence for RNA interference, a more classical role for antisense tRNAs, exists only for tRNA Ser UCN. Mutation pathogenicity probably frequently results from a mixture of effects due to sense and antisense tRNA translational activity for many mitochondrial tRNAs. Genomic studies should routinely explore for translational activity by antisense tRNAs.     

Thio modification of yeast cytosolic tRNA is an iron-sulfur protein-dependent pathway

Defects in the yeast cysteine desulfurase Nfs1 cause a severe impairment in the 2-thio modification of uridine of mitochondrial tRNAs (mt-tRNAs) and cytosolic tRNAs (cy-tRNAs). Nfs1 can also provide the sulfur atoms of the iron-sulfur (Fe/S) clusters generated by the mitochondrial and cytosolic Fe/S cluster assembly machineries, termed ISC and CIA, respectively. Therefore, a key question remains as to whether the biosynthesis of Fe/S clusters is a prerequisite for the 2-thio modification of the tRNAs in both of the subcellular compartments of yeast cells. To elucidate this question, we asked whether mitochondrial ISC and/or cytosolic CIA components besides Nfs1 were involved in the 2-thio modification of these tRNAs. We demonstrate here that the three CIA components, Cfd1, Nbp35, and Cia1, are required for the 2-thio modification of cy-tRNAs but not of mt-tRNAs. Interestingly, the mitochondrial scaffold proteins Isu1 and Isu2 are required for the 2-thio modification of the cy-tRNAs but not of the mt-tRNAs, while mitochondrial Nfs1 is required for both 2-thio modifications. These results clearly indicate that the 2-thio modification of cy-tRNAs is Fe/S protein dependent and thus requires both CIA and ISC machineries but that of mt-tRNAs is Fe/S cluster independent and does not require key mitochondrial ISC components except for Nfs1.