Nuclearencoded plastidial crfactors

Specific transcription initiation in bacteria requires a transcription factor (c), which is responsible for promoter recognition and contributes to DNA melting around the initiation site. Most bacterial genomes contain genes for several c-factors recognizing distinct promoters. Bacterial c-factors possess conserved functional regions and are grouped into two families, c70 and c54 (Wösten 1998; Ishi-hama 2000). The c70-factors are furthermore categorized into primary (group 1, essential for cell growth), non-essential primary (group 2), and alternative c-factors (group 3), responsible for recognition of certain promoters in response to environmental signals (Lonetto et al. 1992; Gruber and Bryant 1997). Cyanobacte-ria, the ancestors of plastids, have also multiple c-factors with distinct promoter specificity (Kaneko et al. 1996).

Early on, biochemically purified c-like activities in plant plastids were reported in Chlamydomonas (Surzycki and Shellenbarger 1976), spinach (Lerbs et al. 1983), and mustard (Bülow and Link 1988; Tiller and Link 1993b). Furthermore, immunological evidence for c-like factors was obtained in chloroplast RNA po-lymerase preparations of maize, rice, Chlamydomonas reinhardtii, and Cyanidium caldarium (Troxler et al. 1994). Moreover, multiple nuclear-encoded genes encoding bacterial c70-type factors were identified in the red algae Cyanidium caldarium (CcaA-C; Liu and Troxler 1996; Tanaka et al. 1996) and Cyandioschyzon merolae (CmeSig1-4; Matsuzaki et al. 2004) suggesting specialized promoter recognition as in bacteria. Correspondingly, c-factor families were identified in genomes of land plants such as Arabidopsis (AthSig1-6; Isono et al. 1997b; Tanaka et al. 1997; Fujiwara et al. 2000; Hakimi et al. 2000), mustard (SalSig1-3; Kestermann et al. 1998; Homann and Link 2003), tobacco (NtaSigA1, -A2; Oikawa et al. 2000), rice (OsaSig1-4; Tozawa et al. 1998; Kasai et al. 2004), maize (ZmaSig1-5; Lahiri et al. 1999; Tan and Troxler 1999; Lahiri and Allison 2000), Physcomitrella patens (PpaSig1, -2, -5; Hara et al. 2001a, 2001b; Ichikawa et al. 2004), as well as wheat (TaeSigA; Morikawa et al. 1999), and Sorghum (SbiSig1; Kroll et al. 1999). Interestingly, the genome of the unicellular green algae Chlamydomonas reinhardtii harbors only a single gene encoding a c-factor (CreRpoD; Carter et al. 2004; Bohne et al. 2006). The N-termini of these c-factors show sequences typical for plastid-targeting transit peptides and indeed have been demonstrated to confer plastidial targeting either of GFP-fusion proteins in vivo (Isono et al. 1997b; Tanaka et al. 1997; Kanamaru et al. 1999; Fujiwara et al. 2000; Lahiri and Allison 2000; Oikawa et al. 2000; Hara et al. 2001a) or with radio-labeled proteins in vitro (Kestermann et al. 1998). Surprisingly, targeting of some plant c-factors occurred not only into plastids but also into mitochondria. Alternative splicing of AthSig5 transcripts within intron 1 establishes two initiation methionines (M1 and M2). Shorter peptides starting with M2 showed exclusive GFP targeting into plastids. However, GFP fusion proteins starting with M1 were localized to mitochondria.

RNA analyses revealed that the longer (plastidial) AthSig5 transcripts are exclusively located in flowers, whereas the shorter (mitochondrial) transcripts were detectable in both flower and leaf tissue (Yao et al. 2003). Furthermore, Ath-Sig1::GFP fusion proteins as well are co-localized to both plastids and mitochondria in tobacco protoplast import assays (H. Tandara and K. Liere, unpublished data). Similarly, dual targeting was shown for the maize ZmaSig2B protein by immunological and GFP-fusion protein import studies. Interestingly, Zma-Sig2B was biochemically co-purified with RpoTm, the mitochondrial phage-type RNA polymerase (Beardslee et al. 2002), suggesting a possible role of these mitochondrial localized c-factors in regulation of plant mitochondrial transcription.

Historically, plastidial c-factors were designated either alphabetically or by numbers. Thus, in Arabidopsis SigA, SigB, and SigC (Tanaka et al. 1997) were also named SIG2, SIG1, and SIG3 (Isono et al. 1997b), respectively. In an effort to unify the nomenclature, c-factors sequences were subjected to phylogenetic analyses and distinguished by numbers (http://sfns.u-shizuoka-ken.ac.jp/pctech; Shiina et al. 2005). Higher plant c-factors belong into a monophyletic group (Lysenko 2006). They are related to bacterial primary (group 1) and non-essential primary (group 2) c70-factors. However, none fit into alternative group 3 nor are related to c54-factors. Phylogenetic analyses revealed that plastidial c-factors are split into at least 5 subgroups: Sig1, Sig2, Sig3, Sig5, and Sig6. Interestingly, the monocot and dicot c-factors within the Sig1 and Sig2 groups are located on separate branches. Most sequenced higher plant and moss genomes contain at least one gene for a Sig1-type c-factor. Since the Arabidopsis Sig1 homologues are highly expressed during chloroplast biogenesis, it is assumed that Sig1 represents the principal c-factor in chloroplasts (Tanaka et al. 1997; Kestermann et al. 1998; To-zawa et al. 1998; Kanamaru et al. 1999; Morikawa et al. 1999). Similarly, Sig2, Sig3, and Sig5 genes have been identified in various plant organisms, suggesting a correspondingly important role in plastidial transcription. Conversely, to date Ath-Sig4 is the only Arabidopsis Sig gene without known ortholog in other plants, and in comparison to the other c-factors its transcription is rather low in light-grown plants (Tsunoyama et al. 2002). Supported by the observation that intron sites of AthSig1, AthSig2, AthSig3, AthSig4, and AthSig6 are almost identical (Fujiwara et al. 2000), phylogenetic analysis suggests that the Sig3, AthSig4, and Sig6 groups are related to Sig2 (Shiina et al. 2005; Lysenko 2006). Although closely related, the Sig1 and Sig2 groups possess different number of introns. These c-factors, therefore, may originate from gene duplication events of one or more ancestral genes. Albeit only partially, the Sig5 group seems to be phylogenetically related to the bacterial alternative c-factors (Tsunoyama et al. 2002; Shiina et al. 2005; Lysenko 2006). AtSig4 is suggested to have originated from partly processed transcript of AthSig2, AthSig3, or AthSig6 inserted as cDNA into the genome, since it is the only Sig gene in higher plants that has lost an intron (Lysenko 2006).

Bacterial c70-factors contain three conserved domains involved in binding the core RNA polymerase (domains 2.1 and 3), hydrophobic core formation (2.2), DNA melting (2.3), recognition of the -10 promoter motif (2.4), and recognition of the -35 promoter motif (Section 4.1, 4.2; Wösten 1998; Paget and Helmann 2003).

Since these domains are as well present in all known plastidial c-factors it is to be expected that they are responsible for transcription from c70-type promoters in plastids. However, structural analysis seems not to provide answers if the role of the different plastidial c-factors is to selectively activate promoters and if they possess distinct or overlapping promoter specificities. Based on the phylogenetic analyses one might presume that plastidial c-factors group into general c-factors involved in transcription of standard c70-type promoters and specialized c-factors responsible for recognition of exceptional promoters in response to developmental and/or environmental cues (Shiina et al. 2005; Lysenko 2006).

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