Continuous Primary Sequence Requirements in the 18-Nucleotide Promoter of Dicot Plant Mitochondria

Abstract

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...

The transcription machineries of plant mitochondria and chloroplasts: Composition, function, and regulation

“…As shown by mutational analyses the promoter regions required for successful transcription initiation extend beyond the obviously conserved regions (Hoffmann and Binder, 2002). Comparing 11 promoters from pea, soybean, potato and Oenothera Dombrowski et al (1999) deduced an extended consensus sequence (AAAATATCATAAGAGAAG, 100% conserved positions in bold, transcription initiation site underlined) that consists of three parts: the conserved nonanucleotide motif from −7 to +2, containing the transcription initiation site; the less conserved AT-box, consisting of predominantly adenosine and thymidine bases located through positions −14 to −8; and the positions +3 and +4, where mainly purines are found. Additionally, certain nucleotide identities are required for efficient in vitro transcription initiation in the AT-rich region.…”

“…In vitro capping techniques, in vitro transcription assays and sequence analysis led to the identification of transcription initiation sites and promoter motifs in Zea mays (Mulligan et al, 1988(Mulligan et al, , 1991Rapp et al, 1993;Caoile and Stern, 1997), Zea perennis (Newton et al, 1995), Glycine max (Brown et al, 1991), Pisum sativum (Binder et al, 1995;Giese et al, 1996;Dombrowski et al, 1999;Hoffmann and Binder, 2002), Triticum aestivum , Nicotiana (Lelandais et al, 1996;Edqvist and Bergman, 2002), Solanum tuberosum (Binder et al, 1994;Lizama et al, 1994;Giese et al, 1996;Remacle and Maréchal-Drouard, 1996), Sorghum (Yan and Pring, 1997) and Oenothera berteriana (Binder and Brennicke, 1993). One type of plant mitochondrial promoters is characterized by a consensus sequence motif (CRTA) similar to the YRTA motif of plastid NEP promoters (see reviews in Fey and Maréchal-Drouard, 1999;Binder and Brennicke, 2003;Weihe, 2004).…”

Distant sequences determine 5' end formation of cox3 transcripts in Arabidopsis thaliana ecotype C24

“…The realization of this genetic information occurs in several consecutive steps, which are so far only partially understood. Transcription is initiated at various promoter sequences, some of which have been functionally characterized by in vitro analyses in different plant species (5,6). In dicots, an 18 bp sequence enclosing the transcription initiation site is necessary and sufficient to drive transcription in vitro (6,7).…”

Phage-Type RNA Polymerase RPOTmp Performs Gene-Specific Transcription in Mitochondria of Arabidopsis thaliana

“…Sequences spanning 18 nucleotides, from position 214 to +4 around the TSS, are displayed. This region has been defined by Dombrowski et al (1999) to be required for promoter function in dicot plant mitochondria. The promoters and putative promoters of genes transcribed less actively in rpoTmp were computationally compared with all other known mitochondrial promoters in Arabidopsis to identify hexa-, penta-, or tetranucleotide elements statistically overrepresented in the subset of promoters affected in rpoTmp (see Supplemental Table 1 online).…”

“…Frequently, multiple promoters are active in the upstream region of one gene (Lupold et al, 1999;Kü hn et al, 2005). Using transcriptionally competent mitochondrial extracts, promoter sequences of up to 25 nucleotides comprising the transcription start site have been defined to be required for correct and efficient initiation of transcription in vitro (Rapp and Stern, 1992;Rapp et al, 1993;Caoile and Stern, 1997;Dombrowski et al, 1999). Plant mitochondrial promoter sequences are highly variable, and in contrast with the abundant information on plastid promoters (Hajdukiewicz et al, 1997;Liere et al, 2004;Courtois et al, 2007;Swiatecka-Hagenbruch et al, 2008), there is very little data on the recognition of these promoters by different RNA polymerases.…”