Department of Plant Physiology, Institute of Biology, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany.
Methods Mol Biol. 2022;2363:279-300. doi: 10.1007/978-1-0716-1653-6_19.
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.
了解线粒体转录起始位点和启动子序列对于理解植物线粒体转录起始机制至关重要。转录起始位点可以通过对初级转录物 5' 端的作图来直接确定。本章描述了一种基于下一代测序的方法，用于在拟南芥中进行线粒体基因组范围的转录起始位点作图。与其他旨在确定初级转录物 5' 端的策略一样，该方案利用了只有初级转录物而不是加工转录物被 5'-三磷酸化的特性，并基于此特性，可通过酶促选择进行。然而，它使用了新生转录物，以便（1）与其他隔间相比，提高线粒体的覆盖率，（2）减少 rRNA 和其他背景，以及（3）还捕获快速降解或加工的转录物的初级 5' 端。
