Highly purified pea chloroplast RNA polymerase transcribes both rRNA and mRNA genes.
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, 115, 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-1,5-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 were found to be localized in chloroplasts by an indirect immunofluorescence.
Rajasekhar
V K
VK
Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California, Irvine 92717.
Sun
E
E
Meeker
R
R
Wu
B W
BW
Tewari
K K
KK
D001427:Bacterial Toxins / Q000494:pharmacology; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D002846:Chromatography, Affinity; D004275:DNA, Ribosomal / Q000235:genetics / Q000648:ultrastructure; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D003998:Dicarboxylic Acids / Q000494:pharmacology; D007887:Fabaceae / Q000201:enzymology / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D017343:Genes, Plant; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010946:Plants, Medicinal; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015183:Restriction Mapping; D014158:Transcription, Genetic / Q000187:drug effects
D001427:Bacterial Toxins; D004275:DNA, Ribosomal; D003998:Dicarboxylic Acids; D009943:Organophosphorus Compounds; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; C062221:tagetitoxin; D012321:DNA-Directed RNA Polymerases