An<i>in vitro</i>system for accurate transcription Initiation of chloroplast protein genes

Orozco, Emil M.; Mullet, John E.; Chua, Nam‐Hai

doi:10.1093/nar/13.4.1283

Cited by 103 publications

(72 citation statements)

References 27 publications

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“…Earlier studies have shown that pea and barley chloroplast membrane high-salt extracts accurately transcribe rbcL and psbA promoters (Orozco et al, 1985). In this study, similar extracts were found to transcribe accurately the psbD LRP.…”

Section: Discussionsupporting

confidence: 69%

“…This result is consistent with tagetitoxin's ability to inhibit lightinduced accumulation of psbD transcripts in barley seedlings (Sexton et al, 1990b). Transcription of the psbD LRP required supercoiled templates similar to transcription of other plastid genes (Jolly and Bogorad, 1980;Orozco et al, 1985;Chen and Orozco, 1988).…”

Section: Discussionmentioning

confidence: 99%

“…To test whether barley chloroplast highsalt extracts (Orozco et al, 1985) reading frame is shown at the far right. The most abundant light-induced psbD transcript generated in vivo and in vitro is marked by an arrow.…”

Section: In Vitro Transcription From the Psbd Lrpmentioning

confidence: 99%

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Identification of a sequence-specific DNA binding factor required for transcription of the barley chloroplast blue light-responsive psbD-psbC promoter.

Kim

Mullet

1995

Plant Cell

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The plastid gene psbD encodes the photosystem II reaction center chlorophyll protein D2. psbD is located in a complex operon that includes psbC, psbK, psbl, orf62, and tmG. The operon is transcribed from at least three different promoters. One of the psbD promoters is differentially activated when plants are exposed t o blue light. In this study, the psbD blue light-responsive promoter was accurately transcribed in vitro in high-salt extracts of barley plastids. Transcription required supercoiled templates and was inhibited by tagetitoxin, an inhibitor of plastid transcription. Escherichia coli RNA polymerase did not recognize the psbD light-responsive promoter with the same specificity as plastid RNA polymerase.Deletion analyses demonstrated that sequences between -39 and -68, upstream of the transcription initiation site, were required for transcription of the psbD blue light-responsive promoter. This DNA region is highly conserved among plant species and contains multiple AAG sequences. Gel shift assays and DNase I footprinting experiments demonstrated that the AAG-rich DNA sequence interacts with a sequence-specific DNA binding factor termed AGF. Point mutations in the AAG c i s element decreased binding of AGF and inhibited transcription from the psbD light-responsive promoter. We concluded that AGF is an essential factor required for transcription of the psbD light-responsive promoter.

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Section: Discussionsupporting

confidence: 69%

Section: Discussionmentioning

confidence: 99%

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Identification of a sequence-specific DNA binding factor required for transcription of the barley chloroplast blue light-responsive psbD-psbC promoter.

Kim

Mullet

1995

Plant Cell

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“…In vivo dissection was performed by studying the expression of uidA reporter genes from an ordered set of PrrnP1 promoter derivatives (Staub and Maliga, 1993;Allison and Maliga, 1995). In vitro dissection was performed by measuring transcript accumulation from mini-genes that consist of a PrrnP1 promoter derivative and transcription terminators (Jolly and Bogorad, 1980;Link, 1984;Gruissem and Zurawski, 1985;Orozco et al, 1985). In vivo dissection of the plastid rrn operon promoter indicates that sequences upstream of the conserved Ϫ 35 box are important for promoter function.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2003

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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 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 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.

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“…The transcription of tRNA and mRNA genes was obtained by a soluble RNA polymerase in chloroplast transcription extracts from spinach (Gruissem et al, 1983a;Gruissem and Zurdwski, 1985a, b), mustard (Link, 1984), maize (Orozco et al, 1985), and Euglena (Gruissem et al, 1983 b;Greenberg et al, 1984). The transcription of the rRNA gene was found to be associated with a DNA-protein complex (transcriptionally active chromosome) that could not be solubilized (Greenberg et al, 1984).…”

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confidence: 99%

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

Rajasekhar

Sun

Meeker

et al. 1991

European Journal of Biochemistry

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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.

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Anin vitrosystem for accurate transcription Initiation of chloroplast protein genes

Cited by 103 publications

References 27 publications

Identification of a sequence-specific DNA binding factor required for transcription of the barley chloroplast blue light-responsive psbD-psbC promoter.

Identification of a sequence-specific DNA binding factor required for transcription of the barley chloroplast blue light-responsive psbD-psbC promoter.

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

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

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