Functional importance of nucleotide identities within the pea atp9 mitochondrial promoter sequence

Sequences ranging from nucleotide positions -14 to +4 relative to the transcription start site constitute an in vitro functional pea atp9 promoter. A comparison of respective sequence segments surrounding 11 unambiguously identified transcription initiation sites of various dicotyledoneous plant species revealed the highest level of evolutionary fidelity of nucleotide identities within the conserved nonanucleotide motif (CNM), suggesting their importance for promoter function. Using a mitochondrial in vitro transcription system, a detailed analysis by site-directed mutagenesis now reveals that the alteration of nucleotides -6 to -2 and +1 within the CNM indeed reduces promoter activity by more than 80%. Changes of nucleotide identities at the less conserved positions -12 to -9 within the AT-rich region reduced the initiation efficiency by about 70%. The alteration of the highly conserved position -7 has little influence on promoter function, indicating that evolutionary conservation does not always correlate with the functional importance of certain nucleotides. Mutagenesis of nucleotides at positions +3 or +4 reveals a minimal requirement of at least one purine for wild-type transcription initiation efficiency. The assignment of functionally important nucleotide identities should now facilitate an efficient and reliable prediction of other promoters in mitochondria of dicotyledon plants.