RESUMEN
Transcripts of typical dicot plant plastid genes undergo C-->U RNA editing at approximately 30 locations, but there is no consensus sequence surrounding the C targets of editing. The cis-acting elements required for editing of the C located at tobacco rpoB editing site II were investigated by introducing translatable chimeric minigenes containing sequence -20 to +6 surrounding the C target of editing. When the -20 to +6 sequence specified by the homologous region present in the black pine chloroplast genome was incorporated, virtually no editing of the transcripts occurred in transgenic tobacco plastids. Nucleotides that differ between the black pine and tobacco sequence were tested for their role in C-->U editing by designing chimeric genes containing one or more of these divergent nucleotides. Surprisingly, the divergent nucleotide that had the strongest negative effect on editing of the minigene transcript was located -20 nt 5' to the C target of editing. Expression of transgene transcripts carrying the 27 nt sequence did not affect the editing extent of the endogenous rpoB transcripts, even though the chimeric transcripts were much more abundant than those of the endogenous gene. In plants carrying a 93 nt rpoB editing site sequence, transgene transcripts accumulated to a level three times greater than transgene transcripts in the plants carrying the 27 nt rpoB editing sites and resulted in editing of the endogenous transcripts from 100 to 50%. Both a lower affinity of the 27 nt site for a trans-acting factor and lower abundance of the transcript could explain why expression of minigene transcripts containing the 27 nt sequence did not affect endogenous editing.
Asunto(s)
Cloroplastos/metabolismo , ADN de Cloroplastos/genética , Edición de ARN , ARN de Planta/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Cloroplastos/genética , ADN Complementario/genética , ADN Complementario/metabolismo , ARN Polimerasas Dirigidas por ADN , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Proteínas de Plantas/genética , Plantas Tóxicas , ARN de Planta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Nicotiana/genética , Transcripción Genética , Transformación GenéticaRESUMEN
Two cysteinyl-tRNA synthetases (CysRS) and four asparaginyl-tRNA synthetases (AsnRS) from Arabidopsis thaliana were characterized from genome sequence data, EST sequences, and RACE sequences. For one CysRS and one AsnRS, sequence alignments and prediction programs suggested the presence of an N-terminal organellar targeting peptide. Transient expression of these putative targeting sequences joined to jellyfish green fluorescent protein (GFP) demonstrated that both presequences can efficiently dual-target GFP to mitochondria and plastids. The other CysRS and AsnRSs lack targeting sequences and presumably aminoacylate cytosolic tRNAs. Phylogenetic analysis suggests that the four AsnRSs evolved by repeated duplication of a gene transferred from an ancestral plastid and that the CysRSs also arose by duplication of a transferred organelle gene (possibly mitochondrial). These case histories are the best examples to date of capture of organellar aminoacyl-tRNA synthetases by the cytosolic protein synthesis machinery.