Recent in vitro studies of the yeast mitochondrial transcription machinery unexpectedly revealed promoter specificity to be conferred by the core RNAP rather than mtTFB (Matsunaga and Jaehning 2004). Similarly, in vitro transcription assays with recombinant AthRpoTm and AthRpoTp enzymes showed accurate initiation of transcription from overlapping subsets of mitochondrial and plastidial promoters without auxiliary factors, therefore retaining a characteristic feature of the T7 RNAP. However, AthRpoTm and AthRpoTp failed to recognize some of the investigated promoters and AthRpoTmp displayed no significant promoter specificity while showing high non-specific transcription activity. Therefore, it is evident that the Arabidopsis enzymes need auxiliary factors for transcription in organello like the mitochondrial RNA polymerases of other organisms (Kühn et al. 2007).
Thus far, identification of factors involved in specific promoter recognition and transcription initiation by NEP has failed. Based on information on such factors interacting with the related mitochondrial phage-type RNA polymerases from humans, mice, Xenopus laevis, and yeast one can only speculate. These mitochon-drial transcription complexes consist of a minimum of two components: the catalytic core enzyme (mtRPO, ~ 120-150 kDa), and a specificity factor, which confers promoter recognition (mtTFB, ~ 40-45 kDa). Despite poor overall sequence similarity, it recently has been shown that mtTFB factors belong to a family of RNA-methyltransferases (Falkenberg et al. 2002; McCulloch et al. 2002; Rantanen et al. 2003; Seidel-Rogol et al. 2003). An additional component, which binds the DNA further upstream, enhances mitochondrial transcription in vitro (mtTFA, 20-25 kDa). This DNA-binding protein belongs to the HMG (high mobility group) family and may also facilitate the interaction with other trans-acting factors (reviewed in Jaehning 1993; Shadel and Clayton 1993; Tracy and Stern 1995; Hess and Börner 1999). To date, no functional mtTFA or mtTFB homologues have been isolated from plant mitochondria or plastids, and the presence of such proteins in plant organelles is unclear. BLAST searches of the Arabidopsis genome revealed a TFB-like dimethyladenosine transferase gene, which possesses an N-terminal transit peptide mediating protein import into plastids of isolated tobacco protoplasts (B. Kuhla, K. Liere, T. Börner; unpublished data). This gene corresponds to the previously characterized PFC1 gene encoding a plastid 16S rRNA dimethylase homologous to the yeast nucleolar 18S rRNA dimethylase Dim1 (Tokuhisa et al. 1998). The phenotype of PFC1-knockout mutants, however, does not support the idea that this TFB-like dimethyladenosine transferase may act as a primary transcription factor for the phage-type RNA polymerases (M. Swiatecka-Hagenbruch, K. Liere, T. Börner, unpublished data).
A good candidate for an activating factor for NEP transcription in spinach is CDF2, which has been reported to stimulate transcription of the rrn operon Pc promoter by NEP-2, a yet to be characterized nuclear-encoded transcription activity (Table 3; Bligny et al. 2000). CDF2 is supposed to exist in two distinct forms, CDF2-A and CDF2-B. CDF2-A might repress transcription initiation of PEP at the rrn16 P1 promoter (termed P2 in spinach), while CDF2-B possibly binds NEP-2 and initiates specific transcription from the rrn16 Pc promoter.
Another factor that is discussed to be involved in NEP transcription is the plas-tidial ribosomal protein L4 (RPL4; encoded by the nuclear Rpl4 gene). A role for RPL4 in NEP transcription was proposed, as it co-purifies with the T7-like transcription complex in spinach (Trifa et al. 1998). In prokaryotes the ribosomal protein L4 was shown to have extra-ribosomal functions in transcriptional regulation (Zengel et al. 1980). The spinach and Arabidopsis Rpl4 genes have acquired remarkable 3' extensions during evolutionary transfer to the nuclear genome, which resemble highly acidic C-terminal ends of certain transcription factors. A function for this protein in NEP or PEP transcription, however, has yet to be demonstrated.
Besides, some nucleus-encoded c-factors for the bacterial-type PEP in plastids were found to additionally localize to mitochondria (see Section 4.2.2). So far phage-type RNA polymerases are the sole transcription activity in mitochondria of higher plants. One may speculate that these c-factors may have an additional role in regulating mitochondrial transcription by these RNA polymerases (H. Tandara and K. Liere, unpublished data; Beardslee et al. 2002; Yao et al. 2003). Yet, ex-
perimental data to link the activity of the bacterial-type plastidial c-factors to the phage-type enzymes in mitochondria or plastids are still lacking.
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