Identification of primary transcriptional start sites of mouse mitochondrial DNA: accurate in vitro initiation of both heavy- and light-strand transcripts

Abstract

The major transcriptional control sequences of vertebrate mitochondrial DNA lie within the displacement loop region. Transcription events initiating in the displacement loop sequence of the mouse genome were identified by 5' end mapping of primary transcripts by S1 nuclease protection and primer extension techniques. Light-strand transcription initiates at a single site, 165 nucleotides upstream of the major heavy-strand origin of replication. Transcription of the heavy strand occurs at two distinct sites, 5 and 13 nucleotides upstream of the gene for phenylalanyl-tRNA, the first heavy-strand-encoded gene. This spatial relationship of the two transcriptional start sites with each other and with the origin of heavy-strand replication and the gene for tRNAPhe is quite similar to that for human mitochondrial DNA. The predominant form of primary heavy-strand transcript in mouse is a short, ca. 75-nucleotide, RNA containing the sequences of tRNAPhe and a few additional nucleotides at the 5' end of tRNAPhe, suggesting that the processing of tRNA involves independent cleavages at the 5' and 3' ends of tRNA sequences.