RESUMEN
The chloroplast psbD and psbC genes encode the D2 and CP43 proteins of the photosystem II complex, and they are generally cotranscribed. We report studies on the basic translation process of tobacco psbD-psbC mRNAs using an in vitro translation system from tobacco chloroplasts. The primary transcript has an unusually long 5'-UTR (905 nt). We show that it is translatable. Processing of the 5'-UTR greatly enhances the translation efficiency of the psbD cistron. A striking feature is that psbD and psbC cistrons overlap by 14 nt. Removal of the psbD 5'-UTR plus the start codon and introduction of a premature termination codon in the psbD cistron considerably reduce the translation efficiency of the downstream psbC cistron. These results indicate that translation of the psbC cistron depends largely on that of the upstream psbD cistron and thus shows translational coupling; however, a portion is independently translated. These observations, together with the presence of monocistronic psbC mRNAs, suggest that the psbD and psbC cistrons are translated via multiple processes to produce necessary amounts of D2 and CP43 proteins.
Asunto(s)
Regiones no Traducidas 5' , Cloroplastos/genética , Complejo de Proteína del Fotosistema II/genética , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Cloroplastos/metabolismo , Complejo de Proteína del Fotosistema II/biosíntesis , ARN del Cloroplasto/biosíntesis , ARN del Cloroplasto/química , ARN del Cloroplasto/metabolismo , ARN Mensajero/química , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
The regulation of gene expression is still one of the major issues in modern plant molecular biology. The amount of RNA in a cell is regulated by both transcriptional and posttranscriptional events. Methods to determine these steady-state levels of RNAs, such as Northern analysis, ribonuclease protection assay (RPA), and quantitative real-time PCR, do not discriminate between regulation by de novo RNA synthesis and the influence by degradation or stabilization. To assess the rate of transcription of individual genes, run-on transcription is utilized. To this end, isolated chloroplasts are used in brief in vitro transcription reactions in the presence of radiolabeled nucleotides, with a subsequent hybridization of the isolated RNA with DNA fragments spotted on membranes. Here, we describe a protocol for run-on transcription in chloroplasts isolated from Arabidopsis leaves and present data on the transcriptional activity of several plastid genes in detached leaves of different Arabidopsis ecotypes.
Asunto(s)
Arabidopsis/genética , Cloroplastos/genética , Técnicas Genéticas , ARN del Cloroplasto/biosíntesis , Transcripción Genética , Regulación de la Expresión Génica de las Plantas , Genes del Cloroplasto/genética , Membranas Artificiales , Hibridación de Ácido Nucleico , ARN del Cloroplasto/genéticaRESUMEN
The RNA editing processes in chloroplasts and mitochondira of higher plants show several similarities which are suggestive of common components and/or biochemical steps between the two plant organelles. The existence of various promiscuous DNA fragments of chloroplast origin in plant mitochondrial genomes allowed us to test the possibility that chloroplast sequences are also edited in mitochondria. An rpoB fragment transferred from chloroplasts to mitochondria in rice was chosen as it contains several editing sites, two of which match sequence motifs surrounding even non-homologous editing sites in both chloroplast and mitochondrial transcripts. Rice chloroplast and mitochondrial rpoB DNA and cDNA sequences were selectively amplified and the editing status of the cDNA sequences was determined. Three of the four potential rpoB editing sites previously detected in maize were found to be edited in the rice chloroplast rpoB transcript, whereas the fourth was found to remain unedited. In mitochondria, however, all four editing sites remain unmodified at the cDNA level. This indicates that the editing processes of higher plant mitochondria and chloroplasts are not identical and that organelle-specific factors are required for eliciting the respective editing events.
Asunto(s)
Cloroplastos/metabolismo , ADN de Cloroplastos/metabolismo , Mitocondrias/metabolismo , Oryza/metabolismo , Proteínas de Plantas/biosíntesis , Edición de ARN , ARN del Cloroplasto/biosíntesis , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Cartilla de ADN , Sondas de ADN , ADN Mitocondrial/química , ADN Mitocondrial/metabolismo , ARN Polimerasas Dirigidas por ADN , Datos de Secuencia Molecular , Proteínas de Plantas/química , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
We report the characterization of transcripts from the halophyte, Mesembryanthemum crystallinum, encoding a protein with high homology to chloroplast RNA-binding proteins (cRBP). In this plant chloroplast-related functions are largely protected against salt stress. cRBP transcripts are derived from a single gene, Mc32crbp, although three size classes of polyadenylated mRNAs are detected. Transcription rate and steady state amounts of mRNA are developmentally regulated and light controlled with strong transcriptional activity as functional chloroplasts are established, and with lower maintenance activity thereafter. Upon salt stress, the rate of transcription decreases, although transcript levels increase. Accompanying stress, a change in the distribution of transcript size classes is observed as the longest transcript with an untranslated 3' end of 381 nucleotides increases relative to transcripts with shorter 3' ends. The long transcript is characterized by the presence of five sequence elements in the 3'-untranslated region that are present in cRBP mRNAs from a variety of plants, although not all elements are found in each mRNA. The results may indicate a mechanism by which mRNA levels of constitutively light-regulated genes may be modulated without enhanced transcription in response to environmental cues.