The maize mitochondrial cox2 gene has five promoters in two genomic regions, including a complex promoter consisting of seven overlapping units

Plant mitochondrial genes are often transcribed into complex sets of RNAs, resulting from multiple initiation sites and processing steps. To elucidate the role of initiation in generating the more than 10 cox2 transcripts found in maize mitochondria, we surveyed sequences upstream of cox2 for active promoters. Because the cox2 coding region is immediately downstream of a 0.7-kb recombination repeat, cox2 is under the control of two different sets of potential expression signals. Using an in vitro transcription assay, we localized four promoters upstream of the coding region in the so-called master chromosome, and two promoters upstream of the coding region in the recombinant subgenome, Ribonuclease protection analysis of labeled primary transcripts confirmed that all but one of these promoters is active in vivo. Primer extension was used to identify the promoter sequences and initiation sites, which agree with the consensus established earlier for maize mitochondria. This study identified two unusual promoters, the core sequences of which were composed entirely of adenines and thymines, and one of which was a complex promoter consisting of seven overlapping units. Deletion mutagenesis of the complex promoter suggested that each of its units was recognized independently by RNA polymerase, While each active promoter fit the maize core consensus sequence YRTAT, not all such sequences surveyed supported initiation. We conclude that in vitro transcription is a powerful tool for locating mitochondrial promoters and that, in the case of cox2, promoter multiplicity contributes strongly to transcript complexity.