Mitochondrial genomes generally encode a minimal set of tRNAs necessary for protein synthesis. However, number eukaryotes import from the cytoplasm into their mitochondria. For instance, Saccharomyces cerevisiae imports cytoplasmic tRNA Gln mitochondrion without any added factors. Here, we examine existence similar active system in mammalian We have used subcellular RNA fractions rat liver and human cells to perform RT-PCR with oligonucleotide primers specific nucleus-encoded CUG UUG...

PCR amplification of template DNAs extracted from mixed, naturally occurring microbial populations, using oligonucleotide primers complementary to highly conserved sequences, was used obtain a large collection diverse RNase P RNA-encoding genes. An alignment these sequences in comparative analysis RNA secondary and tertiary structure. The new confirm the structure model based on cultivated organisms (with minor alterations helices P12 P18), providing additional support for nearly every base...

Aminoacyl-tRNA (aa-tRNA) formation, an essential process in protein biosynthesis, is generally achieved by direct attachment of amino acid to tRNA the aa-tRNA synthetases. An exception Gln-tRNA synthesis, which eukaryotes catalyzed glutaminyl-tRNA synthetase (GlnRS), while most bacteria, archaea, and chloroplasts employ transamidation pathway, a tRNA-dependent glutamate modification generates Gln-tRNA. Mitochondrial synthesis carried out normally mitochondrial enzymes organelle-encoded tRNAs...

Adenosine-to-inosine editing in the anticodon of tRNAs is essential for viability. Enzymes mediating tRNA adenosine deamination bacteria and yeast contain cytidine deaminase-conserved motifs, suggesting an evolutionary link between two reactions. In trypanosomatids, undergo both cytidine-to-uridine adenosine-to-inosine editing, but relationship reactions unclear. Here we show that down-regulation Trypanosoma brucei tRNA-editing enzyme by RNAi leads to a reduction C-to-U A-to-I vivo....

Transfer RNA modifications play pivotal roles in protein synthesis. N6-threonylcarbamoyladenosine (t6A) and its derivatives are found at position 37, 3΄-adjacent to the anticodon, tRNAs responsible for ANN codons. These universally conserved all domains of life. t6A have pleiotropic functions synthesis including aminoacylation, decoding translocation. We previously discovered a cyclic form (ct6A) as chemically labile derivative from bacteria, fungi, plants protists. Here, we report...

Abstract Every type of nucleic acid in cells undergoes programmed chemical post-transcriptional modification. Generally, modification enzymes use substrates derived from intracellular metabolism, one exception is queuine (q)/queuosine (Q), which eukaryotes obtain their environment; made by bacteria and ultimately taken into eukaryotic via currently unknown transport systems. Here, we a combination molecular, cell biology biophysical approaches to show that Trypanosoma brucei tRNA Q levels...

Abstract Transfer RNAs (tRNAs) are key players in protein synthesis. To be fully active, tRNAs undergo extensive post-transcriptional modifications, including queuosine (Q), a hypermodified 7-deaza-guanosine present the anticodon of several bacteria and eukarya. Here, molecular biochemical approaches revealed that protozoan parasite Trypanosoma brucei, Q-containing have preference for U-ending codons asparagine, aspartate, tyrosine histidine, analogous to what has been described other...

The essential deamination of adenosine A

Editing of tRNAs is widespread in nature and either changes the decoding properties or restores folding a tRNA. Unlike phylogenetically disperse adenosine (A) to inosine (I) editing, cytosine (C) uridine (U) editing has only been previously described organellar tRNAs. We have shown that cytoplasmic tRNA(Thr)(AGU) undergoes two distinct events anticodon loop: C U A I. In vivo, every inosine-containing tRNA(Thr) also edited at position 32. vitro, stimulates conversion I wobble base. Although...

All mitochondrial tRNAs in kinetoplastid protozoa are encoded the nucleus and imported from cytosol. Incubation of two vitro-transcribed tRNAs, tRNAIle(UAU) tRNAGln(CUG), with isolated mitochondria Leishmania tarentolae, absence any added cytosolic fraction, resulted a protease-sensitive, ATP-dependent importation, as measured by nuclease protection. Evidence that protection represents importation was obtained finding Bacillus subtilis pre-tRNAAsp protected digestion also cleaved an...

Human mitochondrial tRNA (hmt-tRNA) mutations are associated with a variety of diseases including myopathies, diabetes, encephalopathies, and deafness. Because the current understanding precise molecular mechanisms these is limited, there no efficient method to treat their diseases. Here, we use known in hmt-tRNA(Phe) investigate that lead malfunctions. We tested impact on aminoacylation, structure, translation elongation-factor binding. The majority mutants were pleiotropic, exhibiting...

Fe/S clusters are part of the active site many enzymes and essential for cell viability. In eukaryotes cysteine desulfurase Nfs (IscS) donates sulfur during cluster assembly was thought sufficient this reaction. Moreover, is indispensable tRNA thiolation, a modification generally required function protein synthesis. Recently, Isd11 discovered as an integral activity at early step assembly. Here we show, using combination genetic, molecular, biochemical approaches, that Isd11, in line with...

Due to a complete lack of the tRNA genes in mitochondrial genome Trypanosoma brucei, all tRNAs needed for translation have be imported into organelle from cytosol. A previous study showed that modified nucleotide s(2)U could act as negative determinant import another kinetoplastid, Leishmania tarentolae. We investigated whether same type cytosolic control retention exists T. brucei. Based on Northern analysis with subcellular RNA fractions and vitro assays, we demonstrate silencing cysteine...

Retrograde transport of tRNAs from the cytoplasm to nucleus was first described in Saccharomyces cerevisiae and most recently mammalian systems. Although function retrograde is not completely clear, it plays a role cellular response changes nutrient availability. Under low conditions are sent presumably remain storage there until levels improve. However, S. tRNA constitutive occurs even when adequate. Constitutive important, at least, for proper maturation tRNAPhe, which undergoes...

All tRNAs undergo post-transcriptional chemical modifications as part of their natural maturation pathway. Some modifications, especially those in the anticodon loop, play important functions translational efficiency and fidelity. Among these, 1-methylguanosine, at position 37 (m(1)G37) loop several tRNAs, is evolutionarily conserved participates reading frame maintenance. In eukaryotes, tRNA methyltransferase TRM5 responsible for m(1)G formation nucleus-encoded well mitochondria-encoded...

Establishment of the early genetic code likely required strategies to ensure translational accuracy and inevitably involved tRNA post-transcriptional modifications. One such modification, wybutosine/wyosine is crucial for fidelity in Archaea Eukarya; yet it does not occur Bacteria has never been described mitochondria. Here, we present genetic, molecular mass spectromery data demonstrating first example wyosine mitochondria, a situation thus far unique kinetoplastids. We also show that these...

All mitochondrial tRNAs in <i>Leishmania tarentolae</i> are encoded the nuclear genome and imported into mitochondrion from cytosol. One tRNA (tRNA^Trp) is edited by a C to U modification at first position of anticodon. To determine <i>in vivo</i>substrates for importation as well editing, we examined subcellular localization extent 5′- 3′-end maturation tRNA^Trp(CCA), tRNA^Ile(UAU), tRNA^Gln(CUG), tRNA^Lys(UUU), tRNA^Val(CAC)....

Editing of adenosine (A) to inosine (I) at the first anticodon position in tRNA is catalyzed by deaminases acting on (ADATs). This essential reaction bacteria and eukarya permits a single decode multiple codons. Bacterial ADATa homodimer with two bound Zn(2+). The crystal structure revealed residues important for substrate binding catalysis; however, such high resolution structural information not available eukaryotic deaminases. Despite significant sequence similarity among deaminases, we...

ABSTRACT We describe here the isolation and characterization of a novel RNA-binding protein, RBP38, from Leishmania tarentolae mitochondria. This protein does not contain any known motifs is highly conserved among trypanosomatids, but no homologues were found in other organisms. Recombinant LtRBP38 binds single double-stranded (ds) RNA substrates with dissociation constants 100 nM range, as determined by fluorescence polarization analysis. Downregulation expression homologous gene, TbRBP38,...

To establish the relationships of lizard- and mammal-infecting Leishmania, we characterized intergenic spacer region ribosomal RNA genes from L. tarentolae hoogstraali. The organization these regions is similar to those other eukaryotes. was approximately 4 kb in 5.5 size difference due a greater number 63-bp repetitive elements latter species. This also contained another element, repeated twice, that had an inverted octanucleotide with potential form stem-loop structure could be involved...