RESUMEN
The mitochondrion of the parasitic protozoon Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by partial import of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single copy genes, suggesting the use of the same enzyme in the cytosol and in the mitochondrion. However, the T. brucei genome encodes two distinct genes for eukaryotic aspartyl-tRNA synthetase (AspRS), although the cell has a single tRNAAsp isoacceptor only. Phylogenetic analysis showed that the two T. brucei AspRSs evolved from a duplication early in kinetoplastid evolution and also revealed that eight other major duplications of AspRS occurred in the eukaryotic domain. RNA interference analysis established that both Tb-AspRS1 and Tb-AspRS2 are essential for growth and required for cytosolic and mitochondrial Asp-tRNAAsp formation, respectively. In vitro charging assays demonstrated that the mitochondrial Tb-AspRS2 aminoacylates both cytosolic and mitochondrial tRNAAsp, whereas the cytosolic Tb-AspRS1 selectively recognizes cytosolic but not mitochondrial tRNAAsp. This indicates that cytosolic and mitochondrial tRNAAsp, although derived from the same nuclear gene, are physically different, most likely due to a mitochondria-specific nucleotide modification. Mitochondrial Tb-AspRS2 defines a novel group of eukaryotic AspRSs with an expanded substrate specificity that are restricted to trypanosomatids and therefore may be exploited as a novel drug target.
Asunto(s)
Aspartato-ARNt Ligasa/genética , Aspartato-ARNt Ligasa/metabolismo , ARN de Transferencia de Aspártico/biosíntesis , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genética , Adenosina Trifosfato/metabolismo , Animales , Citosol/enzimología , Diseño de Fármacos , Mitocondrias/enzimología , Filogenia , Especificidad por Sustrato , Aminoacilación de ARN de Transferencia/fisiología , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
The aspartate identity of tRNA for AspRS from Thermus thermophilus has been investigated by kinetic analysis of the aspartylation reaction of different tRNA molecules and their variants as well as of tRNAPhe variants with transplanted aspartate identity elements. It is shown that G10, G34, U35, C36, C38, and G73 determine recognition and aspartylation of yeast and T.thermophilus tRNA(Asp) by the thermophilic AspRS. This set of nucleotides specifies also tRNA aspartylation in the homologous yeast and Escherichia coli systems. Structural considerations indicate that the major aspartate identity elements interact with amino acids conserved in all AspRSs. It follows that the structural features of tRNA and synthetase specifying aspartylation are mainly conserved in various structural contexts and in organisms adapted to different life conditions. Mutations of tRNA identity elements provoke drastic losses of charging in the heterologous system involving yeast tRNA(Asp) and T. thermophilus AspRS. In the homologous systems, the mutational effects are less pronounced. However, effects in E. coli and T. thermophilus exceed those in yeast which are particularly moderate, indicating variations in the individual contributions of identity elements for aspartylation in prokaryotes and eukaryotes. Analysis of multiple tRNA mutants reveals cooperativity between the cluster of determinants of the anticodon loop and the additional determinants G10 and G73 for efficient aspartylation in the thermophilic system, suggesting that conformational changes trigger formation of the functional tRNA/synthetase complex.
Asunto(s)
Aspartato-ARNt Ligasa/metabolismo , ARN de Transferencia de Aspártico/metabolismo , Thermus thermophilus/enzimología , Secuencia de Aminoácidos , Anticodón , Aspartato-ARNt Ligasa/química , Secuencia de Bases , Escherichia coli , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN de Transferencia de Aspártico/biosíntesis , ARN de Transferencia de Fenilalanina/biosíntesis , ARN de Transferencia de Fenilalanina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
To identify the gene coding for the endonuclease which processes the 3' end of mitochondrial (mt) tRNA transcripts in Saccharomyces cerevisiae, nuclear mutations able to complement a mt mutant (Ts932) defective for this process were isolated and analyzed. One of these mutants exhibited a growth defect both on respiratory and fermentable media. Complementation of this phenotype with a S. cerevisiae centrometric wild-type genomic library has allowed us to identify a new essential S. cerevisiae gene strongly conserved in various eukaryotic organisms.
Asunto(s)
Evolución Biológica , Secuencia Conservada , Genes Fúngicos , ARN de Transferencia de Aspártico/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis/genética , ADN Mitocondrial/metabolismo , Humanos , Datos de Secuencia Molecular , Muridae , ARN de Transferencia de Aspártico/biosíntesis , Saccharomyces cerevisiae/crecimiento & desarrollo , Schizosaccharomyces/genética , Homología de Secuencia de AminoácidoRESUMEN
The enzymes tRNA guanine-transglycosylase (Tgt) and S-adenosylmethionine :tRNA ribosyltransferase-isomerase (QueA) participate in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q) in Escherichia coli. Here we show by HPLC analysis and gel retardation that both enzymes interact with an in vitro transcribed tRNA(ASP) from yeast, specifically modified with a Q precursor molecule. RNase I footprinting experiments showed strong protein tRNA contacts in the anticodon stem-loop and a minor interaction with the dihydrouridine loop. This suggests that all identity elements for the recognition of Q-specific tRNAs are clustered in the anticodon region and explains earlier results that both enzymes accept a RNA microhelix with the sequence of an anticodon stem-loop as substrate.