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We have determined transcriptional initiation sites for the ATPase subunit 9 gene on the yeast mitochondrial genome. Using S1 nuclease mapping, in vitro capping of primary transcripts with GTP and guanylyl transferase, and in vitro transcription analysis with purified mitochondrial RNA polymerase, we find the major site of transcriptional initiation to be at a point 630 nucleotides upstream of the coding region for the gene. In addition, we find much lower levels of initiation at a second site 78 nucleotides downstream of the first. Both initiation sites occur at the same position within a nonanucleotide sequence which we have previously found associated with initiation of rRNA synthesis. This work further supports the notion that this nonanucleotide sequence is an integral component of mitochondrial promoters and indicates that the same RNA polymerase is used for transcription of both mRNA and rRNA in yeast mitochondria.
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