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 complementary 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.The established endosymbiont hypothesis describes mitochondria and chloroplasts as remnants of bacteria-like progenitors, which penetrated the original eukaryote 1.5 and 1 billion years ago (1). The prokaryotic origin of the organellar DNAs implies that cis-elements engaged in the expression of the organellar genetic information might be similar to the respective control structures of modern bacteria. Such a similarity is indeed found in chloroplasts, where several components of the genetic system show a close structural and functional relationship to their prokaryotic counterparts (2). Many chloroplast promoters for instance resemble the Ϫ10/Ϫ35-type promoters of contemporary prokaryotes. These promoters serve as ciselements for a largely chloroplast encoded eubacteria-like RNA polymerase. Another, nuclear encoded polymerase initiates transcription at a second less well described class of promoters (3-6). This latter enzyme is of the bacteriophage-like single subunit type, a class of polymerases that also includes the RNA polymerases in mitochondria of animals, fungi, and plants (7-9). These enzymes bind to promoters in close spatial relationship to transcription initiation sites.In mitochondria of Saccharomyces cerevisiae a 9-base-pair-long highly conserved cis-element is found at each of the about 20 transcription initiation sites. This nonanucleotide motif autonomously supports efficient transcription initiation in vitro, with enhanced promoter activity depending on the presence of a purine at position ϩ2 and a pyrimidine at ϩ3. In Xenopus laevis mitochondria an eight-nucleotide-long cis-element drives bi-directional transcription initiation at two different locations. A 15-bp 1 consen...

In pea mitochondria the rpl5, rpsl4 and cob ORFs are clustered in a unique genomic environment and are cotranscribed into a 4.7-kb primary transcript and several other polycistronic RNAs with sizes between 4.0 and 2.3 kb. All of the larger RNAs terminate at a common 3' end, 52 nucleotides downstream of the cob gene. Transcription is initiated at a promoter about 1.3 kb upstream of the rpl5 start codon. The promoter sequence 5'-AATAAGAGA-3' corresponds to the highly conserved 5'-CRTAAGAGA-3' motif often found in promoters in dicot plants. Functional analysis in a homologous in vitro transcription system showed the pea rpl5 promoter to be active, despite the presence of an altered base in first position of the promoter motif. In Oenothera, in contrast to pea, transcription of the rpl5 gene is driven by a promoter motif that conforms perfectly to the consensus sequence. Double inverted repeats located in the 3' flanking regions of the rpsl4 and cob ORFs in pea were investigated with respect to their possible role in defining transcript termini and their potential function in controlling exo- and endonucleolytic processing or transcript stabilization.

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