Chapter title |
Mitochondrial Run-On Transcription Assay Using Biotin Labeling.
|
---|---|
Chapter number | 3 |
Book title |
Plant Mitochondria
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2639-8_3 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2638-1, 978-1-4939-2639-8
|
Authors |
Kühn, Kristina, Kristina Kühn |
Editors |
James Whelan, Monika W. Murcha |
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. |
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