In vitro analysis of the pea chloroplast 16S rRNA gene promoter.

Abstract

A cloned pea chloroplast 16S rRNA gene promoter has been characterized in detail by use of a homologous in vitro transcription system that contains a highly purified chloroplast RNA polymerase. The in vivo and in vitro 16S rRNA transcriptional start site has been identified to be a T on the plus strand, 158 bases upstream of the mature 5' end of the gene. BAL 31 deletions of the 16S rRNA leader region demonstrated that the bases between -66 to +30 relative to the transcriptional start site (+ 1) are necessary for specific 16S transcription. Disruption of canonical TTGACA or TATAAT elements within this region caused complete transcriptional inactivation and prevented protein binding. The topological requirement for 16S transcription was examined by using a construct that synthesized a transcript from the 16S promoter and released it from a pea plastid putative terminator sequence. This minigene was relaxed in vitro with a topoisomerase I from pea chloroplast. It was shown that the 16S promoter was most active when the miniigene plasmid was supercoiled.Chloroplast DNA in higher plants contains rRNA, tRNAs, and about 60 to 80 genes coding for chloroplast-specific proteins. The transcription of these genes has been studied by obtaining crude homologous or heterologous RNA polymerase preparations. A great deal of information on the regulatory sequences for the transcription of tRNA genes and several protein genes has been obtained by using deletions, substitutions, and point mutations in recombinant DNA molecules and in vitro RNA transcription systems (2, 8, 11, 13-15, 20, 24, 27, 30, 31, 33). The chloroplast promoters of tRNA and protein genes contain two elements upstream of their transcriptional start sites that bear significant homology with the TTGACA (-35) and TATAAT (-10) consensus regions of bacterial promoters (3,5,9,16,18).The chloroplast 16S (ctl6S) rRNA gene transcription regulatory sequences have not yet been functionally identified. ctl6S transcription start sites have been mapped in vitro in maize (35) and spinach (22). From these studies, there appear to be regions with sequence homology to bacteral promoter consensus elements upstream of the 16S rRNA mature start site. Whether these elements actually regulate 16S transcription has not been shown. For example, the 16S rRNA leader sequence of spinach plastids contain two consensus promoter regions, only one of which seems to be used in vivo (4, 22).We have previously reported that a highly purified pea chloroplast RNA (ctRNA) polymerase preparation was able to faithfully initiate and synthesize the full-length pea 16S rRNA from supercoiled recombinant DNA (36). The in vitro start site was located by S1 nuclease analysis to several nucleotides 155 to 158 bases upstream of the 5' end of the coding region of the mature 16S rRNA. In this paper, we further report that the highly purified ctRNA polymerase can utilize linearized plasmid DNA to produce runoff transcripts. Using this technique coupled with capping of the RNA, we have identified the transcripti...

Unique Architecture of the Plastid Ribosomal RNA Operon Promoter Recognized by the Multisubunit RNA Polymerase in Tobacco and Other Higher Plants

“…Preliminary analysis of the PrrnP1 promoter has been reported in pea (Sun et al, 1989). Because transcription was performed with linear DNA, which is a poor template for the PEP, the pea data are not directly comparable with our results.…”

“…Transcription of the plastid rRNA operon ( rrn ) in higher plants is from diverse promoters. The rrn operon in tobacco is transcribed by the multisubunit, plastid-encoded RNA polymerase (PEP) from a 70 -type promoter (PrrnP1) (Vera and Sugiura, 1995), as in most higher plants, including maize (Strittmatter et al, 1985), pea (Sun et al, 1989), carrot (Manna et al, 1994), rice (Silhavy and Maliga, 1998), barley (Hubschmann and Borner, 1998), and Arabidopsis (Sriraman et al, 1998a). In tobacco, in addition to the PrrnP1 PEP promoter, rrn is transcribed from a second promoter, PrrnP2, which is recognized by the nucleus-encoded plastid RNA polymerase (Vera and Sugiura, 1995;Allison et al, 1996).…”

Organ-specific Transcription of the rrn Operon in Spinach Plastids

“…Taking into account that rrn transcription is 50 to 80 times lower in root than in leaf tissues we used much higher protein concentrations to analyze root extracts compared with leaf extracts. The formation of the CDF2-promoter complex is detectable using leaf extracts (lanes [1][2][3][4][5][6]. No protein/promoter interaction is detected with root plastid extracts even if the protein concentration was scaled up to 52 times that of cotyledon extracts (lanes 9 and 12).…”

“…4 l (lanes 2, 5, 8, and 11) and 8 l (lanes 3, 6, 9, and 12) of 80,000 ϫ g supernatant of lysed tobacco leaf chloroplasts were incubated with the wild-type spinach plastid rrn promoter region (lanes 2 and 3), mutation 3 (lanes 5 and 6), mutation 2 (lanes 8 and 9), and mutation 1 (lanes 11 and 12). Lanes 1,4,7, and 10 represent the labeled DNA fragments without protein.…”

“…These E. coli-like consensus sequences serve as promoter structures (3)(4)(5) or as regulatory elements (6,7). However, the interpretation of results on studies of transcriptional regulation in plastids is complicated by the existence of different types of RNA polymerases.…”