Plastidial Promoters NEP promoters

Unambiguous identification of transcription initiation sites for a nuclear-encoded transcription activity (i.e. NEP) became feasible in plants with reduced or elimi-

nated transcriptional activity by PEP. Such systems comprise the ribosome-deficient plastids of the monocot albostrians barley and iojap maize mutants (Hübschmann and Börner 1998; Silhavy and Maliga 1998a), tobacco Arpo plants (Allison et al. 1996; Hajdukiewicz et al. 1997; Serino and Maliga 1998), Arabi-dopsis lacking PEP due to the action of spectinomycin which blocks plastidial protein synthesis (Swiatecka-Hagenbruch et al. 2007), and photosynthetically inactive tobacco and rice suspension cultures, with elevated levels of NEP activity (Vera et al. 1996; Kapoor et al. 1997; Miyagi et al. 1998; Silhavy and Maliga 1998b).

Most non-photosynthetic genes involved in housekeeping functions such as transcription and translation have promoters for both RNA polymerases NEP and PEP. NEP transcripts of these genes are, with a few exceptions, rarely detectable in chloroplasts and were therefore mostly analyzed in PEP-deficient plants (see above). Only a few genes are known to be transcribed exclusively from a NEP promoter, i.e. accD, encoding a subunit of the acetyl-CoA carboxylase in dicots; ycf2, encoding a protein with a yet unknown function; rpl23, encoding a ribosomal protein; clpP, encoding the proteolytic subunit of the Clp ATP-dependent protease, in monocots; and, most interestingly, the rpoB operon encoding three of the four PEP core subunits in all higher plants (Hajdukiewicz et al. 1997; Hübschmann and Börner 1998; Silhavy and Maliga 1998a; Swiatecka-Hagenbruch et al. 2007). Consequently, PEP abundance and activity depends on the nuclear-encoded RNA polymerase.

NEP promoters analyzed thus far resemble mitochondrial and phage promoters in their structural organization. Based on their sequence properties they can be grouped into three types (Fig. 4; Weihe and Börner 1999; Liere and Maliga 2001). Type-I promoters are characterized by a conserved YRTa-motif critical for rpoB promoter recognition embedded in a small DNA fragment (-15 to +5) upstream of the transcription initiation site (+1) (PatpB-289; Kapoor and Sugiura 1999; Xie and Allison 2002; PaccD-129; Liere and Maliga 1999b; PrpoB-345; Liere and Maliga 1999a). Transient expression of chimeric Arabidopsis rpoB 5'-flanking re-gion::GUS deletion-constructs in cultured tobacco cells suggested upstream regulatory regions for rpoB expression (Inada et al. 1997). However, no additional sequence elements outside the promoter core altered rpoB transcription in vitro (Liere and Maliga 1999a). Similar transient transcription assays to examine the 5'-flanking region of the tobacco accD gene revealed putative sequence elements up-and downstream of the promoter to determine its strength (Hirata et al. 2004). A subset of Type-I NEP promoters possesses a second conserved sequence motif (ATAN0-1GAA) ~18 to 20 bp upstream of the YRTa-motif, designated box II or GAA-box (Fig. 4; Silhavy and Maliga 1998a; Kapoor and Sugiura 1999). Muta-tional analyses of the tobacco PatpB-289 promoter in in vitro and in vivo transcription experiments suggested a functional role of this element in promoter recognition (Kapoor and Sugiura 1999; Xie and Allison 2002). Hence, Type-I promoters are grouped into two subgroups: Ia, with only the YRTa-motif, and Ib, carrying both YRTa- and GAA-box (Weihe and Börner 1999; Liere and Börner 2007).

Pep And Nep

Fig. 4. Schematic overview of different types of PEP and NEP promoters. PEP promoter: the wheat psbA (TaepsbA), barley psbD BLRP (HvupsbD), tobacco rrn16 (Ntarrn16), and the tobacco rbcL PEP promoters (NtarbcL) are shown. Conserved -10/-35 consensus elements, as well as individual promoter elements as the TATA-box (Eisermann et al. 1990), extended -10 sequence (TGn; Satoh et al. 1999), AAG-box (Kim et al. 1999), RUA-element (Suzuki et al. 2003), and RLBP-binding region (Kim et al. 2002) are indicated. The less conserved -35 element in the barley psbD BRLP is shown in grey. NEP promoter: typical architectures of Type-I, Type-II, and Pc NEP promoters from tobacco and spinach are shown with their names in brackets. The YRTa promoter core and GAA-box are marked (Hübschmann and Börner 1998; Kapoor and Sugiura 1999; Liere and Maliga 1999). TIS: transcription initiation site, indicated by arrows. The -35 and -10 elements not used in spinach rrn16 promoter recognition are shown in grey.

Fig. 4. Schematic overview of different types of PEP and NEP promoters. PEP promoter: the wheat psbA (TaepsbA), barley psbD BLRP (HvupsbD), tobacco rrn16 (Ntarrn16), and the tobacco rbcL PEP promoters (NtarbcL) are shown. Conserved -10/-35 consensus elements, as well as individual promoter elements as the TATA-box (Eisermann et al. 1990), extended -10 sequence (TGn; Satoh et al. 1999), AAG-box (Kim et al. 1999), RUA-element (Suzuki et al. 2003), and RLBP-binding region (Kim et al. 2002) are indicated. The less conserved -35 element in the barley psbD BRLP is shown in grey. NEP promoter: typical architectures of Type-I, Type-II, and Pc NEP promoters from tobacco and spinach are shown with their names in brackets. The YRTa promoter core and GAA-box are marked (Hübschmann and Börner 1998; Kapoor and Sugiura 1999; Liere and Maliga 1999). TIS: transcription initiation site, indicated by arrows. The -35 and -10 elements not used in spinach rrn16 promoter recognition are shown in grey.

Type-II NEP promoters lack the YRTa-motif and differ completely in sequence and organization from Type-I promoters. So far this class is represented by a single example, a promoter of the ClpP protease subunit gene (Fig. 4). The tobacco PclpP-53 was characterized using a transplastomic in vivo approach demonstrating that critical promoter sequences are located mainly downstream of the transcription initiation site (-5 to +25; Sriraman et al. 1998a). The clpP-53 promoter motif and transcription initiation site are conserved among monocots, dicots, conifers, and liverworts. But, although present, the tobacco PclpP-53 sequence motif is not used as a promoter in rice and Chlamydomonas. If the rice sequence is introduced into tobacco plastids, the tobacco NEP recognizes this conserved Type-II promoter. Therefore, the lack of transcription in rice from the PclpP-53 homologue may be resulting from either the lack of a Type-II specificity factor or to the lack of a distinct NEP enzyme not present in monocots (e.g. RpoTmp, see Chapter 1.1; Sriraman et al. 1998a; Liere et al. 2004). However, experimental data supporting one or the other of these scenarios are still missing.

Another non-YRTa-type promoter, which is attributed to be recognized by a NEP transcription activity is the rrn operon Pc promoter in spinach and Arabidop-

sis (Fig. 4, 6; Pc promoter; Baeza et al. 1991; Iratni et al. 1994, 1997; Sriraman et al. 1998a; Swiatecka-Hagenbruch et al. 2007). In spinach, the Pc promoter solely drives rrn operon transcription. Although it contains typical a70-elements which are active as the rrn operon promoter in other species, transcription initiates from a site between the conserved -10/-35 hexamers. However, sequences relevant for transcription initiation from Pc have yet to be identified.

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