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...

Pea chloroplast RNA polymerase has been obtained with about 2000-fold purification using DEAE-cellulose and phosphocellulose chromatography. The purified enzyme contained ten prominent polypeptides of 150, 130, 11 5,110,95,85,75,48,44 and 39 kDa and four other minor polypeptides of 90,34,32 and 27 kDa. Purification of this enzyme using chloroplast 16s rDNA promoter affinity column chromatography also yielded an enzyme with similar polypeptides. Purified polyclonal antibodies against the purified chloroplast RNA polymerase were found to recognize most of the polypeptides of the enzyme in Western blot experiments. Primary mobility shift of the 16s rRNA gene and ribulose-l,5-bisphosphate carboxylase large subunit (rbc-L) gene promoters observed with the chloroplast RNA polymerase was abolished by these antibodies. The specific in vitro transcription of these rRNA and mRNA genes was also inhibited by these antibodies. The transcription of the rRNA and mRNA genes was also abolished by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase. The chloroplast RNA polymerase was found to bind specifically to the chloroplast 16s rRNA gene promoter region as visualized in electron microscopy. The presence of the polypeptides of 130, 110, 75-95 and 48 kDa in the DNA-enzyme complex was confirmed by a novel approach using immunogold labeling with the respective antibodies. The polypeptides of this purified RNA polymerase immunofluorescence.

A highly purified RNA polymerase preparation from pea chloroplasts has been shown to specifically transcribe the 16S rRNA gene in vitro using the recombinant pCB2-8 DNA as a template. The RNA polymerase has been found to show maximum activity and specificity with pea supercoiled rDNA as a template. At low concentrations of ribonucleoside triphosphates, the RNA polymerase selectively initiates transcription on the 16S rRNA gene. A part of the 16S rRNA gene has been sequenced. The mature 16S rRNA has been found by S1 nuclease analysis to contain sequences starting from GAAGCT. The in vitro synthesized RNA has been found to protect the same nucleotides GAAGCT. In addition, the in vitro synthesized RNA was also found to strongly protect bases starting with TATG located at about 260 bases away from the start site of the mature 16S rRNA.

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 minigene plasmid was supercoiled.