PEP plastidencoded plastid RNA polymerase

The chromosomes of plastids from nearly all plants contain genes for core sub-units of PEP, a bacterial-type RNA polymerase, which might be responsible for transcription of all plastidial genes in algae but shares responsibility for plastid transcription with one or more NEP enzymes in higher plants (see above). The rpoA gene codes for the 38-kDa a-subunit of PEP (Little and Hallick 1988; Ruf and Kössel 1988; Hu and Bogorad 1990). Like in bacteria, it forms an operon together with several ribosomal protein-encoding genes (Purton and Gray 1989). The Physcomitrella plastome lacks this gene. Instead, rpoA is found in the nuclear genome (Sugiura et al. 2003). A similar situation was reported for the bacterial-type RNA polymerase in chloroplasts of several algae (Smith and Purton 2002) and in the plastid-like organelles (apicoplasts) of Plasmodium (Wilson et al. 1996). The ß- (120 kDa), ß'- (85 kDa), and ß"-subunits (185 kDa) are encoded by the rpoB, rpoC1, and rpoC2 genes, respectively, which together form an operon, exactly as known from cyanobacteria (Ohyama et al. 1986; Hudson et al. 1988; Little and Hallick 1988; Hu et al. 1991; Kaneko et al. 1996; Shinozaki et al. 1986; reviewed in Lysenko and Kuznetsov 2005). The structural relationship of the E. coli RNA polymerase and PEP was confirmed by reconstituting a functional E.

coli enzyme with polypeptides truncated as in PEP (Severinov et al. 1996). The high degree of conservation kept by PEP during evolution from the bacterial RNA polymerase is also demonstrated by its sensitivity to tagetitoxin (e.g. Mathews and Durbin 1990; Sakai et al. 1998). Other potent inhibitors of transcription in bacteria, rifampicin, and its related drugs, were also shown to inhibit transcription by the E. coli-like form of PEP found in etioplasts but not by the more complex form in chloroplasts (Fig. 1; e.g. Surzycki 1969; Loiseaux et al. 1975; Pfannschmidt and Link 1997). Furthermore, replacing the PEP a-subunit with the E. coli homologue in transplastomic tobacco resulted in a non-functional PEP enzyme, indicating that the evolutionary conservation of both a-subunits is insufficient to allow such an exchange (Suzuki and Maliga 2000).

The rpoBC operon is under control of a NEP promoter in monocotyledonous and dicotyledonous plants (see Section 3.1). Transcript levels of rpo genes are low compared with genes for proteins involved in photosynthesis (e.g. Hess et al. 1993; Legen et al. 2002). For promoter recognition, the core subunits have to be complemented by a sigma (ct) factor. Sigma factors are encoded by nuclear genes in all embryophytes (see Section 4.2.2) ensuring together with NEP a control of plastid transcription by the nucleus.

While NEP activity (demonstrated by recognition of NEP promoters) could be found hitherto only in soluble fractions of plastid lysates, PEP can be isolated from plastids as a 'soluble' (DNA-dependent) enzyme and in a 'insoluble' (DNA-associated) form together with DNA and other proteins of unknown function as the so-called 'transcriptionally active chromosome' (TAC; e.g. Briat et al. 1979; Greenberg et al. 1984; Little and Hallick 1988; Suck et al. 1996; Krause and Krupinska 2000; Pfalz et al. 2006). In the case of Euglena, the soluble RNA polymerase fraction and TAC were reported to transcribe different sets of genes. If this is due to the presence of different RNA polymerases, as discussed by Little and Hallick (1988), different transcriptions factors, or has other reasons is unclear yet (Smith and Purton 2002). The soluble PEP fraction contains different proteins and exhibits different sensitivity against rifampicin if prepared from etioplasts vs. chloroplasts. PEP isolated from etioplasts of mustard seedlings consists mainly of the core subunits (Pfannschmidt and Link 1997), whereas PEP preparations from chloroplasts were found to be more complex and contain additional proteins that might be needed for transcription and regulation of transcription under the conditions of light and active photosynthesis (Pfannschmidt and Link 1994, 1997; Link 1996; Baginsky et al. 1999; Pfannschmidt et al. 2000; Ogrzewalla et al. 2002) as discussed below.

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