RESUMO
In plant mitochondria, some of the tRNAs are encoded by the mitochondrial genome and resemble their prokaryotic counterparts, whereas the remaining tRNAs are encoded by the nuclear genome and imported from the cytosol. Generally, mitochondrial isoacceptor tRNAs all have the same genetic origin. One known exception to this rule is the group of tRNA(Gly) isoacceptors in dicotyledonous plants. A mitochondrion-encoded tRNA(Gly) and at least one nucleus-encoded tRNA(Gly) coexist in the mitochondria of these plants, and both are required to allow translation of all four GGN glycine codons. We have taken advantage of this atypical situation to address the problem of tRNA/aminoacyl-tRNA synthetase coevolution in plants. In this work, we show that two different nucleus-encoded glycyl-tRNA synthetases (GlyRSs) are imported into Arabidopsis thaliana and Phaseolus vulgaris mitochondria. The first one, GlyRS-1, is similar to human or yeast glycyl-tRNA synthetase, whereas the second, GlyRS-2, is similar to Escherichia coli glycyl-tRNA synthetase. Both enzymes are dual targeted, GlyRS-1 to mitochondria and to the cytosol and GlyRS-2 to mitochondria and chloroplasts. Unexpectedly, GlyRS-1 seems to be active in the cytosol but inactive in mitochondrial fractions, whereas GlyRS-2 is likely to glycylate both the organelle-encoded tRNA(Gly) and the imported tRNA(Gly) present in mitochondria.
Assuntos
Arabidopsis/enzimologia , Fabaceae/enzimologia , Glicina-tRNA Ligase/metabolismo , Plantas Medicinais , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Cromatografia Líquida , DNA de Plantas , Escherichia coli/genética , Glicina-tRNA Ligase/genética , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Frações Subcelulares/enzimologia , Especificidade por SubstratoRESUMO
The yeast Abc1 protein acts as a chaperone-like protein essential for the proper conformation and efficient functioning of the respiratory complex III. By functional complementation of a yeast abc1 mutant, we have identified an Arabidopsis thaliana cDNA that corresponds to a single copy gene and encodes a protein sharing 45% similarity with the yeast Abc1p protein. Cytochrome spectra and respiratory activity measurements have shown that the plant protein allows a partial restoration of the complex III activity. No major difference in the steady-state level of ABC1At mRNA was observed in various plant tissues, suggesting that ABC1At is constitutively expressed in A. thaliana. Phylogenetic analysis revealed that the Abc1At protein belongs to a large family of proteins composed of two eukaryotic and one prokaryotic subgroups differing by their degree of similarity and probably by their function.
Assuntos
Arabidopsis/genética , DNA Complementar/isolamento & purificação , Complexo III da Cadeia de Transporte de Elétrons/genética , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , DNA Complementar/genética , Deleção de Genes , Dosagem de Genes , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Filogenia , Proteínas de Plantas/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Bean nuclear genes for tRNA(Pro), tRNA(Thr) and tRNA(Leu) were isolated. Expression of the tRNA(Pro) genes was demonstrated in vivo and sequence analysis suggested amplification of the tRNA(Pro) gene copy number through duplication of a gene cluster at the same locus of the bean genome. The two tRNA(Thr) genes isolated were actively transcribed and their transcripts processed in a HeLa cell system. In vivo expression tests of these genes and aminoacylation assays of the corresponding in vitro transcripts showed the presence of identity determinants in the anticodon of plant tRNA(Thr). The tRNA(Leu) gene was not expressed due to deviation from the consensus in the internal B-box promoter. The same sequence deviation also prevented aminoacylation of the corresponding in vitro transcript. This tRNA(Leu) however exists in plants and is synthesized from another gene with a consensus B-box promoter. Plant mitochondria import from the cytosol a number of nucleus-encoded tRNAs, including tRNA(Leu) and tRNA(Thr). From the available sequence data, we could not identify any conserved structural motif characteristic for the nucleus-encoded tRNAs imported into plant mitochondria, either in the tRNAs, or in the gene flanking sequences. These results suggest that recognition of tRNAs for import is idiosyncratic and likely to depend on protein/RNA interactions that are specific to each tRNA or each isoacceptor group.
Assuntos
Fabaceae/genética , Mitocôndrias/genética , Plantas Medicinais , RNA de Transferência/genética , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Fabaceae/metabolismo , Regulação da Expressão Gênica de Plantas , Células HeLa , Humanos , Dados de Sequência Molecular , RNA de Transferência/isolamento & purificação , RNA de Transferência/metabolismo , RNA de Transferência de Leucina/biossíntese , RNA de Transferência de Leucina/isolamento & purificação , RNA de Transferência de Leucina/metabolismo , RNA de Transferência de Prolina/biossíntese , RNA de Transferência de Prolina/isolamento & purificação , RNA de Transferência de Prolina/metabolismo , RNA de Transferência de Treonina/biossíntese , RNA de Transferência de Treonina/isolamento & purificação , RNA de Transferência de Treonina/metabolismoRESUMO
To study tRNA import into plant mitochondria, we have set up a system to follow the fate in vivo of tRNA transcripts introduced into plant protoplasts by electroporation. Conditions were optimized for maximum tRNA uptake into potato protoplasts. We have shown that in vitro synthesized tRNA transcripts are poor substrates due to rapid degradation leading to low efficiencies of transfer and short life in protoplasts. Labelled natural tRNAs were more efficiently electroporated into protoplasts and they remained stable during protoplast culture. We have observed import into mitochondria of total and purified cytosolic tRNAs in protoplasts but the process was not specific for the tRNA species which are normally imported.