Unique architecture of the plastid ribosomal RNA operon promoter recognized by the multisubunit RNA polymerase in tobacco and other higher plants

Abstract

Expression of the plastid rRNA operon (rrn) during development is highly regulated at the level of transcription. The plastid rrn operon in most higher plants is transcribed by the plastid-encoded RNA polymerase (PEP), the multisubunit plastid RNA polymerase from PrrnP1, a sigma(70)-type promoter with conserved -10 and -35 core promoter elements. To identify functionally important sequences, the tobacco PrrnP1 was dissected in vivo and in vitro. Based on in vivo deletion analysis, sequences upstream of nucleotide -83 do not significantly contribute to promoter function. The in vitro analyses identified an essential hexameric sequence upstream of the -35 element (GTGGGA; the rRNA operon upstream activator [RUA]) that is conserved in monocot and dicot species and suggested that the -10 element plays only a limited role in PrrnP1 recognition. Mutations in the initial transcribed sequence (+9 to +14) enhanced transcription, the characteristic of strong promoters in prokaryotes. We propose that sigma interaction with the -10 element in PrrnP1 is replaced in part by direct PEP-RUA (protein-DNA) interaction or by protein-protein interaction between the PEP and an RUA binding transcription factor.

Figure 1.

Inspection of the Tobacco Plastid trnV-rrn Intergenic Region for Potential Regulatory Elements. Alignment of the tobacco plastid (Nt) and E. coli (Ec) rRNA operon upstream regions. Nucleotide position is given relative to the transcription initiation site (+1; horizontal arrows). The Fis binding sites (boldface) and the promoter recognition region, including the UP element and the conserved −35 and −10 elements, are marked. For tobacco, the 3′ end of trnV gene also is indicated.

Figure 2.

Identification of PrrnP1 Upstream Promoter Elements by Testing uidA Transcript Accumulation in Vivo. (A) Tobacco PrrnP1 promoter deletion derivatives. Nucleotide position is given relative to the transcription initiation site (+1; horizontal arrows). The positions of the conserved −35 and −10 elements are marked. Plasmid names are listed at left. (B) Plastid transformation vector with left and right targeting sequences, the selectable marker (aadA), and the uidA reporter gene. The positions of plastid genes rrn16, trnV, and rps12/7 and the relevant restriction sites are indicated. Horizontal arrows indicate gene orientation. (C) RNA gel blot to test the steady state levels of uidA mRNA in transplastomic plants. Probing for the cytoplasmic 25S rRNA was used as a loading control. WT, wild type.

Figure 3.

Identification of PrrnP1 Upstream Promoter Elements in Vitro. (A) Construct for testing in vitro promoter activity. Arrows t₁ and t₂ denote the two transcripts terminating within the his (This) and thr (Tthr) attenuators. (B) Tobacco PrrnP1 promoter deletion derivatives. Nucleotide position is given relative to the transcription initiation site (+1; horizontal arrows). The positions of the conserved −35 and −10 elements are marked. Plasmid names are listed at right. (C) Autoradiograph of the in vitro transcripts, and relative quantities. Values were determined as described in Methods and are averages of three experiments.

Figure 4.

Scanning Mutagenesis to Map PrrnP1 Promoter Elements in Vitro. (A) DNA sequences of Prrn promoter derivatives. Plasmid names and mutated nucleotide positions are listed at left. At top, a horizontal arrow marks the positions of multiple transcription initiation sites (TIS), and vertical arrowheads mark the −64, +17, and +37 positions relative to the transcription initiation site marked +1. Relevant cloning sites are labeled. The conserved −35 and −10 promoter elements are boxed. Dots in the alignment represent identical sequences. Nonplastid nucleotides are shown in lowercase, and mutated nucleotides are shown in boldface. (B) Autoradiograph of the in vitro transcripts, and relative quantities. The origin of the t₁ and t₂ transcripts is explained in the legend to Figure 3. Signals of transcripts derived from the three initiation sites do not resolve. Bars represent the sum of signals of the t₁ plus t₂ transcripts relative to clone pJYS112 (100%). This figure was obtained by merging two independently obtained data sets. Values for plasmids pJYS15 through pJYS124 and plasmids pJYS112 through pJYS183 were normalized to their own control (pJYS112 [100%]; black bar). Values were determined as described in Methods and are averages of three experiments.

Figure 5.

Probing −10 Function in Vitro. (A) DNA sequences of wild-type and mutant −10 regions in barley (Kim et al., 1999) and tobacco (this study) plastid promoters. (B) Autoradiograph of the in vitro transcripts, and relative transcription activity of the tobacco PrrnP1 derivatives. Values were determined as described in Methods and are averages of three experiments.

Figure 6.

Model for the Interaction of the PEP with the PrrnP1 Promoter. (A) Factor-independent activation of PrrnB1 transcription. Depicted is the σ interaction with the RUA −35 region. Note that the actual subunit involved in the interaction may be a different subunit. Identified are the α- (αCTD and αNTD), β-, β′-, β′′-, and σ-subunits and the RUA, −35, and −10 promoter elements. (B) Factor-dependent activation of PrrnP1 transcription by activator bound to RUA.

Figure 7.

Alignment of the trnV and rrn Intergenic Region in the Plastid Genome of Monocot and Dicot Species. Data are shown for tobacco (Nt), rice (Os), maize (Zm), spinach (So), carrot (Dc), Arabidopsis (At), soybean (Gm), and pea (Ps). The ends of the structural genes for trnV and rrn are bracketed. The RUA, −35, and −10 conserved promoter elements are boxed. Horizontal arrows mark transcription initiation sites from the Pc, PrrnP1 (P1), and PrrnP2 (P2) promoters. Vertical arrows denote the positions of tobacco processing sites. Dashes represent gaps in the alignment. Conserved nucleotide positions are denoted by asterisks below the alignment.