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PubMed1993

Transposition of a group II intron.

Among mobile genetic elements, self-splicing introns are of particular interest. They belong to either group I or group II depending on their three-dimensional structure. Homing, the systematic intron invasion of an intronless gene when it encounters its homologous intron-bearing allele, is the only means for intron mobility so far demonstrated. It depends on the activity of the intron-encoded protein and is very specific for the acceptor site. Intron transposition, the transfer of an intron to a novel site, predicted on the basis of phylogenetic studies and in vitro reverse-splicing experiments, has been proposed to be responsible for evolutionary intron spreading. Here we present results from polymerase chain reaction experiments consistent with transposition of a group II intron. This event is proposed to account for the site-specific deletion in the mitochondrial chromosome of the fungus Podospora anserina that is associated with the premature death syndrome and might also be involved in the senescence process affecting this species.

Ascomycota↗
PubMed2001

Human cells are protected from mitochondrial dysfunction by complementation of DNA products in fused mitochondria.

Extensive complementation between fused mitochondria is indicated by recombination of 'parental' mitochondrial (mt) DNA (ref. 1,2) of yeast and plant cells. It has been difficult, however, to demonstrate the occurrence of complementation between fused mitochondria in mammalian species through the presence of recombinant mtDNA molecules, because sequence of mtDNA throughout an individual tends to be uniform owing to its strictly maternal inheritance. We isolated two types of respiration-deficient cell lines, with pathogenic mutations in mitochondrial tRNAIle or tRNALeu(UUR) genes from patients with mitochondrial diseases. The coexistence of their mitochondria within hybrids restored their normal morphology and respiratory enzyme activity by 10-14 days after fusion, indicating the presence of an extensive and continuous exchange of genetic contents between the mitochondria. This complementation between fused mitochondria may represent a defence of highly oxidative organelles against mitochondrial dysfunction caused by the accumulation of mtDNA lesions with age.

DNA, Mitochondrial↗
PubMed1992

RNA binding determinant in some class I tRNA synthetases identified by alignment-guided mutagenesis.

The N-terminal nucleotide binding folds of all 10 class I tRNA synthetases (RSs) contain characteristic conserved sequence motifs that define this class of synthetases. Sequences of C-terminal domains, which in some cases are known to interact with anticodons, are divergent. In the 676-amino acid Escherichia coli methionyl-tRNA synthetase (MetRS), interactions with the methionine tRNA anticodon are sensitive to substitutions at a specific location on the surface of the C-terminal domain of this protein of known three-dimensional structure. Although four class I synthetases of heterogeneous lengths and unknown structures are believed to be historically related to MetRS, pair-wise sequence similarities in the region of this RNA binding determinant are obscure. A multiple alignment of all sequences of three of these synthetases with all MetRS sequences suggested a location for the functional analog of the anticodon-binding site in these enzymes. We chose a member of this set for alignment-guided mutagenesis, combined with a functional analysis of mutant proteins. Substitutions within two amino acids of the site fixed by the multiple sequence alignment severely affected interactions with tRNA but not with ATP or amino acid. Multiple individual replacements at this location do not disrupt enzyme stability, indicating this segment is on the surface, as in the MetRS structure. The results suggest the location of an RNA binding determinant in each of these three synthetases of unknown structure.

Diphosphates↗
PubMed1989

Construction of intra-domain chimeras of aminoacyl-tRNA synthetases.

To explore new approaches to enzyme engineering, intra-domain chimeras of two aminoacyl-tRNA synthetases were constructed. Connections were made within the nucleotide folds of these enzymes at sites earlier shown either to be dispensable for activity or able to accommodate oligopeptide insertions. (R.M. Starzyk, T.A. Webster and P. Schimmel, Science 237, 1614 (1987); R.M. Starzyk, J.J. Burbaum and P. Schimmel, Biochemistry, in press). Based on the known structure of one synthetase and structural modeling of the other, the locations of the connection sites allow the possibility of functional "compound" ATP and tRNA binding sites. Of five chimeric genes which were constructed, three direct synthesis of polypeptides that accumulate in vivo. These stable hybrids provide prototypes to which mutagenesis procedures may be applied to produce enzymatically active chimeric synthetases.

Adenosine Triphosphate↗
PubMed2005

tRNA actively shuttles between the nucleus and cytosol in yeast.

Previous evidence suggested that transfer RNAs (tRNAs) cross the nuclear envelope to the cytosol only once after maturing in the nucleus. We now present evidence for nuclear import of tRNAs in yeast. Several export mutants accumulate mature tRNAs in the nucleus even in the absence of transcription. Import requires energy but not the Ran cycle. These results indicate that tRNAs shuttle between the nucleus and cytosol.

Active Transport, Cell Nucleus↗
PubMed1997

Discrete determinants in transfer RNA for editing and aminoacylation.

During translation errors of aminoacylation are corrected in editing reactions which ensure that an amino acid is stably attached to its corresponding transfer RNA (tRNA). Previous studies have not shown whether the tRNA nucleotides needed for effecting translational editing are the same as or distinct from those required for aminoacylation, but several considerations have suggested that they are the same. Here, designed tRNAs that are highly active for aminoacylation but are not active in translational editing are presented. The editing reaction can be controlled by manipulation of nucleotides at the corner of the L-shaped tRNA. In contrast, these manipulations do not affect aminoacylation. These results demonstrate the segregation of nucleotide determinants for the editing and aminoacylation functions of tRNA.

Acetylation↗
PubMed1995

Switching recognition of two tRNA synthetases with an amino acid swap in a designed peptide.

The genetic code is based on specific interactions between transfer RNA (tRNA) synthetases and their cognate tRNAs. The anticodons for methionine and isoleucine tRNAs differ by a single nucleotide, and changing this nucleotide in an isoleucine tRNA is sufficient to change aminoacylation specificity to methionine. Results of combinatorial mutagenesis of an anticodon-binding-helix loop peptide were used to design a hybrid sequence composed of amino acid residues from methionyl- and isoleucyl-tRNA synthetases. When the hybrid sequence was transplanted into isoleucyl-tRNA synthetase, active enzyme was generated in vivo and in vitro. The transplanted peptide did not confer function to methionyl-tRNA synthetase, but the substitution of a single amino acid within the transplanted peptide conferred methionylation and prevented isoleucylation. Thus, the swap of a single amino acid in the transplanted peptide switches specificity between anticodons that differ by one nucleotide.

Amino Acid Sequence↗
PubMed1992

Mitochondrial DNA mutations in cardiomyopathy.

Deletions and point mutations of mitochondrial DNA (mtDNA) of patients with dilated or hypertrophic cardiomyopathy were analyzed using the polymerase chain reaction and fluorescence-based direct sequencing. The patients included are with hypertrophic cardiomyopathy associated with left ventricular dilatation, a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and a patient with fatal infantile cardiomyopathy. Deletions were frequently seen in mtDNA in patients with dilated cardiomyopathy. The mtDNA was sequenced and the direct repeat at each edge of deletion was identified as (5'-CATCAACAACCG-3') which was located in the ATPase6 gene and in the D-loop region. In a patient with hypertrophic cardiomyopathy associated with left ventricular dilatation, another mutant mtDNA was found not to have directly repeated sequence, and was revealed to jump from nucleotide position 8,992 to position 16,072 of mtDNA resulting in a 7,079 bp deletion. This patient had unique point mutation in the tRNA genes. A G-to-A transition in the tRNA(Cys) gene (nucleotide position 5,821) at the aminoacyl acceptor stem and an A-to-G transition in the tRNA(Thr) gene (nucleotide position 15,951) were identified. In a patient with MELAS, an A-to-G transition in the tRNA(Leu)(UUR) gene (nucleotide position 3,243) was observed. This mutation was located at the 5' end of the dihydrouridine loop of this tRNA molecule, and would disturb its function. In a patient with hypertrophic cardiomyopathy associated with lactic acidosis, mutations of mtDNA should be suspected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Identification and mapping of trnI, trnE and trnfM genes in the sunflower mitochondrial genome.

Three sunflower mitochondrial HindIII restriction fragments containing the tRNA genes trnI, trnE and trnfM have been sequenced. The genes are present in single copy on the whole genome and are transcribed. Hybridization experiments and sequence analysis of the HindIII fragments allowed the precise mapping and orientation of each gene on the sunflower mitochondrial genome.

Base Sequence↗
PubMed2000

An aminoacylation-dependent nuclear tRNA export pathway in yeast.

Yeast Los1p, the homolog of human exportin-t, mediates nuclear export of tRNA. Using fluorescence in situ hybridization, we could show that the export of some intronless tRNA species is not detectably affected by the disruption of LOS1. To find other factors that facilitate tRNA export, we performed a suppressor screen of a synthetically lethal los1 mutant and identified the essential translation elongation factor eEF-1A. Mutations in eEF-1A impaired nuclear export of all tRNAs tested, which included both spliced and intronless species. An even stronger defect in nuclear exit of tRNA was observed under conditions that inhibited tRNA aminoacylation. In all cases, inhibition of tRNA export led to nucleolar accumulation of mature tRNAs. Our data show that tRNA aminoacylation and eEF-1A are required for efficient nuclear tRNA export in yeast and suggest coordination between the protein translation and the nuclear tRNA processing and transport machineries.

Carrier Proteins↗
PubMed2004

[mtDNA mutations in mouse tumors].

OBJECTIVE: To investigate variations of mtDNA in mouse tumors and to explore the relationship between mtDNA mutations and murine carcinogenesis. METHODS: Variations of D-loop, ND3 and tRNAIle + Glu + Met gene fragments of mtDNA from six mouse tumor cell lines were analyzed by PCR-RFLP and PCR-SSCP techniques. RESULTS: ND3 and tRNAIle + Glu + Met gene fragments of mtDNA from the tumors showed no variations at 27 endonuclease sites. The D-loop of mtDNA from Hca-F demonstrated an additional endonuclease site of Hinf I in contrast to the inbred mouse. Upon PCR-SSCP analysis, the D-loop of mtDNA was found to possess mutations in 4 of 6 tumors. CONCLUSION: D-loop appears to be the hot spot for tumor mtDNA mutations, which may contribute to the carcinogenesis of murine tumors.

Animals↗
PubMed1988

Enzymatic alteration of the anticodon IAC of Torulopsis tRNAVal to IAU for isoleucine anticodon and aminoacylation of the variant.

By enzymatic cleavage and ligation of tRNAVa1, its anticodon sequence IAC was altered to IAU, the anticodon of tRNAI1e. Valine acceptor activity of this variant tRNAVa1 (IAU) was reduced to the extent much lower than tyrosine acceptability of the previously prepared tRNATyr (GAA) (anticodon for tRNAPhe). Isoleucine acceptor activity was undetected, contrary to tRNATyr (GAA) which accepted phenylalanine weakly. Cleavage of tRNAVa1 (IAC) between IACA37 and C38 of its anticodon loop reduced the valine acceptor activity, suggesting some contribution of the conformation of the anticodon loop to the aminoacylation reaction.

Acylation↗
PubMed1994

How the ribosome moves along the mRNA during protein synthesis.

The movement of a ribosome along the mRNA was assessed by the following experimental strategy. mRNAs were synthesized which contained a short coding sequence with at least four codons and a 32P label at one end and an oligo(C) sequence at the other end. When these mRNAs were fixed on the ribosome with tRNAs specific for the defined codons, the oligo(C) stretches were partially outside of the ribosome, whereas the labeled ends were inside the ribosome and thus protected. The overhanging oligo(C) regions were trimmed with the cytidyl-specific RNase CL3 identifying the nucleotides of the mRNAs, which emerged from the ribosome. An mRNA enters the ribosome at nucleotide 18 +/- 1, when counting starts at the first nucleotide of the P-site codon, and leaves the ribosome at nucleotide -21 +/- 2. The ribosome does not move at either side upon A-site occupation but does so at both sides simultaneously upon translocation. The results further indicate that most of the mRNA stretches downstream and upstream of the coding region are not required for the translocation reaction. It is therefore likely that the tRNAs are pulling the mRNA through the ribosome via codon-anticodon interactions in the course of translocation.

Amino Acid Sequence↗
PubMed2006

Differential annotation of tRNA genes with anticodon CAT in bacterial genomes.

We have developed three strategies to discriminate among the three types of tRNA genes with anticodon CAT (tRNA(Ile), elongator tRNA(Met) and initiator tRNA(fMet)) in bacterial genomes. With these strategies, we have classified the tRNA genes from 234 bacterial and several organellar genomes. These sequences, in an aligned or unaligned format, may be used for the identification and annotation of tRNA (CAT) genes in other genomes. The first strategy is based on the position of the problem sequences in a phenogram (a tree-like network), the second on the minimum average number of differences against the tRNA sequences of the three types and the third on the search for the highest score value against the profiles of the three types of tRNA genes. The species with the maximum number of tRNA(fMet) and tRNA(Met) was Photobacterium profundum, whereas the genome of one Escherichia coli strain presented the maximum number of tRNA(Ile) (CAT) genes. This last tRNA gene and tilS, encoding an RNA-modifying enzyme, are not essential in bacteria. The acquisition of a tRNA(Ile) (TAT) gene by Mycoplasma mobile has led to the loss of both the tRNA(Ile) (CAT) and the tilS genes. The new tRNA has appropriated the function of decoding AUA codons.

Anticodon↗