title: Mitochondrial run-on transcription assay using biotin labeling.
abstract: RNA synthesis and different posttranscriptional processes shape the transcriptome of plant mitochondria. It is believed that mitochondrial transcription in plants is not stringently controlled, and that RNA degradation has a major impact on mitochondrial steady-state transcript levels. Nevertheless, the presence of two RNA polymerases with different gene specificities in mitochondria of dicotyledonous species indicates that transcriptional mechanisms may provide a means to control mitochondrial steady-state RNA pools and gene expression. To experimentally assess transcriptional activities in mitochondria, run-on transcription assays have been developed. These assays measure elongation rates for endogenous transcripts in freshly prepared mitochondrial extracts. The mitochondrial run-on transcription protocol described here has been optimized for the model plant Arabidopsis (Arabidopsis thaliana). It uses mitochondria prepared from soil-grown Arabidopsis plants and employs nonradioactive labeling for the subsequent detection of run-on transcripts.
authors.affiliation: Molekulare Zellbiologie der Pflanzen, Institut für Biologie, Humboldt Universität zu Berlin, Philippstraße 13, Berlin, 10115, Germany, kristina.kuehn@hu-berlin.de.
authors.forename: Kristina
authors.initials: K
authors.lastname: Kühn
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000235:genetics*; D019921:Biotinylation; D002458:Cell Fractionation / Q000379:methods; D018506:Gene Expression Regulation, Plant; D008928:Mitochondria / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics*; D014158:Transcription, Genetic; D059467:Transcriptome
chemical_list: D018749:RNA, Plant