Architecture of plastid promoters

So far, only a few plastid promoters have been mapped and analyzed in detail. However, it has become clear that PEP and NEP recognize distinct types of promoter sequences (Liere and Börner, 2007; Liere, et al., 2011). Due to the eubacterial origin of plastids, it is not surprising that the majority of promoters utilized by PEP are similar to E. coli σ70 promoters (Gatenby et Figure 3: RpoT polymerases in organelles of different organisms. Genes in the nucleus (N) encode RpoT polymerases which are targeted to plastids (P) and/or mitochondria (M). In dicots with diploid genomes there are three RpoT genes – RpoTm, RpoTp and RpoTmp; their products are targeted to the mitochondria, plastids and both organelles, respectively. Therefore, NEP activity in dicots can be carried out by two polymerases, RpoTp and RpoTmp. In monocots. NEP is represented by a single RpoTp polymerase. Chlamydomonas possesses only one RpoT gene that is most likely coding for a mitochondria RNA polymerase (mtRNAP). Modified after Liere, et al., 2011.

al., 1981; Gruissem and Zurawski, 1985; Strittmatter et al., 1985). Moreover, E.coli RNA polymerase is able to accurately transcribe from these PEP promoters (Boyer and Mullet, 1986;

Bradley and Gatenby, 1985). PEP promoters consist of -35 (TTGaca) and -10 (TAtaaT) consensus elements (Gatenby, et al., 1981; Gruissem and Zurawski, 1985; Liere and Börner, 2007; Strittmatter, et al., 1985). Some PEP promoters are characterized by additional cis- regulatory sequences. For example, the mustard psbA promoter was shown to have an additional regulatory element (TATA-box) between the -10 and -35 promoter element, which was able in vitro to promote a basal level of transcription without the presence of the -35 region in plastid extracts from dark and light grown plants. Nevertheless, the -35 element was essential for reaching the full promoter activity required during active photosynthesis (Eisermann, et al., 1990; Link, 1984). In the case of the wheat psbA promoter, even though present, the TATA-box seems not to have an important function. Instead, an extended -10 sequence (TGnTATAAT) is utilized as the sole psbA promoter element by PEP isolated from the leaf tip which contains mature chloroplasts. PEP obtained from young plastids in the leaf base, however, still needed both the -10 and -35 boxes (Satoh, et al., 1999). Several cis-elements required for the binding of regulatory proteins have been also described. A twenty-two bp sequence, known as the AAG box, was found to play an important role in the regulation of the blue light-responsive promoter of psbD (coding for the photosystem II reaction center chlorophyll protein D2) by providing the binding site for the AGF (AAG-binding factor) protein complex, which acts as a positive regulator (Kim, et al., 1999). Similarly, the RLPB (rbcL promoter binding) factor was found to enhance transcription upon binding to the sequence -3 to -32 nt upstream of the rbcL transcription start site (Kim, et al., 2002).

1.4.2.2 NEP promoters

In green chloroplasts, PEP transcripts are overrepresented, while most of the transcripts generated by NEP are of low abundance, and are thus rarely detectable (Hess and Börner, 1999;

Liere and Maliga, 2001). Therefore, identification of NEP transcription start sites has been only feasible in plants with knocked out/down PEP activity. Examples of such experimental systems are the albostrians barley and iojap maize mutants which carry ribosome deficient plastids, heterotrophically cultures BY2 cell of tobacco, transplastomic tobacco plants with deleted rpo genes, and Arabidopsis plants grown on spectinomycin which inhibits plastid translation

INTRODUCTION | 11 (Allison, et al., 1996; Hübschmann and Börner, 1998; Serino and Maliga, 1998; Silhavy and Maliga, 1998; Swiatecka-Hagenbruch, et al., 2007; Vera, et al., 1996).

The NEP promoters, analyzed so far, share high sequence similarity with phage and plant mitochondrial promoters (Allison, et al., 1996; Liere and Börner, 2007; Vera, et al., 1996). This is in agreement with the fact that RpoTm alone or together with RpoTmp transcribe the mitochondrial genome of plants and algae (Liere and Börner, 2007). Based on their architecture, NEP promoters can be grouped into three types (Weihe and Börner, 1999; Liere and Maliga, 2001). The majority of analyzed NEP promoters belong to Type-I NEP promoters, which are further divided into two subclasses. Type-Ia promoters consist of a conserved YRTa core motif located shortly upstream of the transcription start site (Liere and Börner, 2007; Liere, et al., 2011). A classical example of a Type-Ia NEP promoter is PrpoB-345 (transcription from this promoter initiates 345 nt upstream of the rpoB ORF in tobacco; Serino and Maliga, 1998).

Deletion analysis of the 5’-flanking region of the Arabidopsis rpoB fused to GUS and transiently expressed in cultured tobacco cells suggested the existence of upstream regulatory elements in addition to the YRTa core (Inada, et al., 1997). On the contrary, no sequence elements outside of the core were found to have a significant influence on the in vitro transcription from the tobacco rpoB promoter (Liere and Maliga, 1999). Type-Ib NEP promoters are characterized by carrying an additional conserved sequence motif (ATAN_0-1GAA), referred to as the GAA-box, that is located approximately 18 to 20 bp upstream of the YRTA motif (Weihe and Börner, 1999; Liere and Börner, 2007). Deletion analysis of the tobacco PatpB-289 promoter revealed a functional role of this element in promoter recognition both in vivo and in vitro (Kapoor and Sugiura, 1999;

Xie and Allison, 2002).

Transcription from Type-II NEP promoters is YRTa independent and is instead controlled by

“non-consensus” promoter elements (Liere, et al., 2011). The most closely investigated example is the tobacco PclpP-53, with a regulatory core sequence found to comprise the region -5 to +25 with respect to the transcription initiation site (Sriraman, et al., 1998). Interestingly, the clpP-53 promoter sequence is conserved among monocots, dicots and C. reinhardtii but it does not drive transcription in rice and Chlamydomonas. However, when introduced into tobacco, the rice PclpP-53 sequence is efficiently utilized, which suggests that this promoter sequence might be recognized by a distinct transcription factor or a NEP enzyme that is present in dicots but not monocots, such as RpoTmp (Liere, et al., 2004; Sriraman, et al., 1998).

The Pc promoter of the rrn operon described in spinach, Arabidopsis and mustard represents another non-YRTa type of NEP promoters (Liere and Börner, 2007). The promoter region of the rrn operon is highly conserved in plants and contains both -10 and -35 PEP promoter elements, which are driving the transcription of the operon in barley, tobacco, maize and in later developmental stages of Arabidopsis (Allison, et al., 1996; Hübschmann and Börner, 1998;

Strittmatter, et al., 1985; Vera and Sugiura, 1995; Courtois, et al., 2007). However, in spinach, as well as during the early development in Arabidopsis, NEP initiates at a site between the conserved PEP elements (Baeza, et al., 1991; Iratni, et al., 1994; Iratni, et al., 1997; Swiatecka-Hagenbruch, et al., 2007; Courtois, et al., 2007).

1.4.2.3 Internal promoters of tRNA genes

The majority of tRNA genes are transcribed by PEP from typical σ⁷⁰-like promoters upstream the transcription start site (Liere and Börner, 2007). However, there are reports suggesting transcription from internal promoters for several tRNA genes, i.e. the spinach trnS, trnR and trnT (Cheng, et al., 1997; Gruissem, et al., 1986), the mustard trnS, trnH and trnR (Liere and Link, 1994; Neuhaus and Link, 1990; Nickelsen and Link, 1990) and the trnE of Chlamydomonas (Jahn, 1992). Furthermore, the coding region of the trnS from spinach alone was shown to be sufficient to promote basal levels (8%) of transcription in in vitro assays (Wu, et al., 1997).

However, the exact features of tRNA-internal promoter elements and the polymerase(s) recognizing them remain to be further elucidated.