Determining mitochondrial transcript termini for the study of transcription start sites and transcript 5′ end maturation

Abstract

Mitochondrial gene expression in plants is considerably more complex than in animals or fungi. In plants, mitochondrial transcripts are generated from transcription initiation at numerous, poorly conserved promoters located throughout the mitochondrial genome. Most genes have more than one transcription start site. Posttranscriptional RNA 5′ end maturation contributes to the diversity of transcripts produced from each mitochondrial gene. Understanding transcriptional mechanisms and transcript maturation requires knowledge on transcription start sites and processing sites. This chapter describes two different, complementary experimental approaches for determining these sites in mitochondrial genomes through mapping of transcript 5′ ends. In order to distinguish 5′ ends deriving from transcription initiation, both strategies exploit the presence of triphosphates at these specifi c 5′ termini.