Abstract
Knowledge of mitochondrial transcription start sites and promoter sequences is key to understanding mechanisms of transcription initiation in plant mitochondria. Transcription start sites can be straightforwardly determined by the mapping of primary transcript 5′ ends. This chapter describes a next-generation sequencing–based protocol for the mitochondrial genome-wide mapping of transcription start sites in Arabidopsis thaliana. Like other strategies aiming at the determination of primary transcript 5′ ends, this protocol exploits that only primary but not processed transcripts are 5′-triphosphorylated and, based on this property, can be enzymatically selected for. However, it uses nascent transcripts, in order to (1) enhance mitochondrial coverage compared with other compartments, (2) reduce rRNA and other background, and (3) also capture the primary 5′ ends of rapidly degraded or processed transcripts.
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Acknowledgments
The work to develop plant organelle nascent transcript sequencing has been supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), grant numbers CRC TRR175-A01 and 400681449/GRK2498-P08.
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Saleh, O., Dwiani, S., Rott, J., Kühn, K. (2022). Nascent Transcript Sequencing for the Mapping of Promoters in Arabidopsis thaliana Mitochondria. In: Van Aken, O., Rasmusson, A.G. (eds) Plant Mitochondria. Methods in Molecular Biology, vol 2363. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1653-6_19
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DOI: https://doi.org/10.1007/978-1-0716-1653-6_19
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