Numerous transcription initiation sites exist for the maize mitochondrial genes for subunit 9 of the ATP synthase and subunit 3 of cytochrome oxidase.

Transcripts for plant mitochondrial genes are frequently present as multiple size classes. In maize, these differences often result from variation in the 5' noncoding region. To determine where transcription initiates, primary (unprocessed) transcripts were specifically labeled in vitro by the capping reaction catalyzed by guanylyltransferase. Direct mapping of transcription initiation sites was accomplished by hybridization of in vitro-capped RNA with the 5' flanking sequences of mitochondrial genes and subsequent digestion with single-strand-specific RNases. The RNase protection experiments identified three transcription initiation sites for subunit 3 of cytochrome oxidase and at least six transcription initiation sites for subunit 9 of ATP synthase. Thus, transcript size heterogeneity is primarily the result of multiple transcription initiation sites for these genes rather than RNA processing. Primer extension analyses of maize mitochondrial RNA were used to precisely establish the sequences at the initiation sites. Comparison of sequences at transcription initiation sites suggests that some homology exists at these sites, although no highly conserved consensus sequence is obvious.