Highly purified pea chloroplast RNA polymerase transcribes both rRNA and mRNA genes

Abstract

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.

Regulation of rDNA transcription in chloroplasts: promoter exclusion by constitutive repression.

“…Very little is known of the processes engaged in transcription and processing. Transcription has been studied by expression of the genes in heterologous systems such as E. coli (Tohdoh et al 1981;Lescure et al 1985;Delp et al 1987) and by in vitro transcription using run-on assays or highly purified chloroplast enzymes (Briat et al 1987;Sun et al 1989;Rajasekhar et al 1991). For maize and pea, it was reported that transcription starts downstream of a -1 0 and -3 5 E. coli-like promoter sequence (Strittmatter et al 1985;Sun et al 1989).…”

Differential Transcription of Pea Chloroplast Genes during Light-Induced Leaf Development (Continuous Far-Red Light Activates Chloroplast Transcription)

“…Transcription of the plastid genes is mediated by at least two RNA polymerases (Greenberg et al, 1984;Rajasekhar et al, 1991;Hess et al, 1993;Lerbs-Mache, 1993) and is modulated by a-like factors (Bülow and Link, 1988;Tiller et al, 1991;Troxler et al, 1994). One of the plastid RNA polymerases is encoded by the plastid genome (rpoA, B, C1, C2).…”

pTAC2, -6, and -12 Are Components of the Transcriptionally Active Plastid Chromosome That Are Required for Plastid Gene Expression

“…The specific activity of the isolated complexes depends on the purification procedure (Suck et al, 1996;. Initially, it was believed that sRNAP transcribes preferentially tRNA genes, while TAC is involved in rRNA transcription (Gruissem et al, 1983;Narita et al, 1985); however, run-on transcription assays uncovered that both highly purified fractions transcribed rRNA, tRNA, and plastid protein-coding genes (Reiss and Link, 1985;Rajashekar et al, 1991;Krupinska and Falk, 1994). Finally, TAC and sRNAP preparations from proplastids, chloroplasts, and etioplasts have different protein compositions, demonstrating their dynamics in response to environmental and developmental changes (Reiss and Link, 1985;Pfannschmidt and Link, 1994;Suck et al, 1996).…”