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, soy bean, 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 complementary nucleotid e identities results in an almost complete loss of promoter activity, sugge sting that primary structure rather than a simple accumulation of adenines and thymidines in this region is essential for promoter activity. This prom oter segment thus appears to be involved in sequence specific binding of a respective protein factor(s) rather than merely loosening and melting the D NA helix during or for an initiation event. Manipulation of nucleotide iden tities in the 3' portion of the pea atp9 promoter and the respective S'-fla nking region revealed that essential sequences extend to positions +3/+4 be yond this transcription start site. Efficient transcription initiation at a n 18-base pair promoter sequence ranging from nucleotide positions -14 to 4 integrated into different sequence contexts shows this element to be suff icient for autonomous promoter function independent of surrounding sequence s.