Continuous primary sequence requirements in the 18-nucleotide promoter of dicot plant mitochondria

The nucleotide requirements of mitochondrial promoters of dicot plants were studied in detail in a pea in vitro transcription system. Deletions in the 5' regions of three different transcription initiation sites from pea, soybean, and Oenothera identified a crucial AT-rich sequence element (AT-Box) comprising nucleotide positions -14 to -9 relative to the first transcribed nucleotide. Transversion of the AT-Box sequence to comple- mentary nucleotide identities results in an almost complete loss of promoter activity, suggesting that primary structure rather than a simple accumulation of adenines and thymidines in this region is essential for promoter activity. This promoter segment thus appears to be involved in sequence specific binding of a respective protein factor(s) rather than merely loosening and melting the DNA helix during or for an initiation event. Manipulation of nucleotide identities in the 3' portion of the pea atp9 promoter and the respective 3'-flanking region revealed that essential sequences extend to positions +3/+4 beyond this transcription start site. Efficient transcription initiation at an 18-base pair promoter sequence ranging from nucleotide positions -14 to +4 integrated into different sequence contexts shows this element to be sufficient for autonomous promoter function independent of surrounding sequences.