Chloroplast RNA polymerase from spinach: purification and DNA-binding proteins

Lerbs, S.; Briat, Jean‐François; Mache, Régis

doi:10.1007/bf01595167

Cited by 59 publications

(31 citation statements)

References 21 publications

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“…It will, however, require further studies to arrive at a definite conclusion, as it is possible that many of these polypeptides are still inseparable contaminants that strongly adhere together. In spinach, the polypeptides of 150,145,110,102,90,85,75,38,33 and 25 kDa were considered prominent in the purified chloroplast RNA polymerase (Lerbs et al, 1983(Lerbs et al, ,1985(Lerbs et al, ,1988. On the other hand in maize, two high-molecular-mass polypeptides of 180 and 140 kDa and twelve other polypeptides of 27-110 kDa constitute the functional chloroplast RNA polymerase (Smith and Bogorad, 1974;Kidd and Bogorad, 1980) and the 180, 120,85 and 38-kDa polypeptides were prominent components (Hu and Bogorad.…”

Section: Discussionmentioning

confidence: 99%

“…There was also a low level of reaction around 165-kDa polypeptide range. With a 4-fold dilution of crude antiserum against native chloroplast RNA polymerase (Lerbs et al, 1983) or purified antibodies of corresponding concentration (600 pg/ml). there was a strong homologous reaction, but the reactions with polypeptides such as the 165-kDa one and other minor polypeptides were not proportionately increased (data not shown).…”

Section: Antibodies To Native Chloroplast R N a Polymerase And Its Vamentioning

confidence: 99%

“…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). There were also some differences in biochemical properties and the polypeptide composition between the soluble and the transcriptionally active chromosome (Briat and Mache, 1980;Lerbs et al, 1983;Narita et al, 1985;Bulow et al, 1987). These data suggested that there may be two different types of RNA polymerases in chloroplasts.…”

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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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Section: Discussionmentioning

confidence: 99%

Section: Antibodies To Native Chloroplast R N a Polymerase And Its Vamentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…One unit of activity is that amount giving 1 pmol UMP incorporation in 10 min. Protein was assayed as described (23). Assays to determine Ki values were as described (29) salience of our noted similarities in amanitin sensitivity of plant and animal polymerases II is that it appears that the amatoxin affinity for polymerase II from these life forms is well conserved through evolution as is the DNA binding site (5 (38), we have consistently seen protoplasts to be less sensitive than walled cells (also in tobacco, J. F. Preston unpublished observations).…”

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confidence: 92%

Sensitivity of Carrot Cell Cultures and RNA Polymerase II to Amatoxins

Little¹,

Preston²

1985

Plant Physiol.

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ABSTRACIProtoplast and cell suspension cultures of Daucus carota L. were evaluated for their sensitivity toward the three amatoxin derivatives, aamanitin, 6'-deoxy-a-amanitin, and 6'-O-methyl-a-amanitin using inhibition of DNA synthesis to measure cell viability. Protoplasts appeared approximately 10-fold more refractory than suspension cells and aamanitin was much less effective than the other two amatoxins, even though Ki values for isolated RNA polymerase II were similar (4-5 nanomolar). Additional studies evaluating the recoveries of all three amatoxins from cell suspension supernates indicate one basis for these differences to be the selective degradation of a-amanitin. A mechanism involving the activation of the hydroxyindole moiety of the a-amanitin is thus invoked to explain these differences and we postulate the involvement of plant oxidases in this role.The establishment by several laboratories that at least three classes of nuclear RNA polymerase are required for DNA-dependent transcription was followed by later demonstrations that plant cells also possess these same classes of enzymes of similar properties (reviewed in Guilfoyle [18]). Contemporary investigations have yielded much basic information on RNA polymerase II, the class of RNA polymerase responsible for transcription ofstructural genes and some small nuclear RNA species (18,43 (37) and Drosophila (15) polymerases II.Although the choices ofplant systems to use for such selections are many, the use of carrot cell suspensions, which readily regenerate through somatic embryogenesis to whole plants, provides several advantages. In addition to simple ploidy (2n = 18) and ease of cultivation, carrot cultures proceed through distinct stages of embryogenesis, providing a unique developmental sequence for study (33). Further, since the appearance embryospecific and callus-specific proteins punctuates this developmental sequence, carrot should be an ideal system to elucidate the molecular bases of plant regeneration (33).Recently, carrot lines have been described as amanitin resistant through a suggested altered sensitivity of RNA polymerase II (38). In our studies directed toward obtaining such lines, we have examined the sensitivity of whole cells and purified polymerase II to two natural and one semisynthetic amatoxin derivatives and show that derivatives in which the 6'-hydroxy moiety is either absent or converted to the corresponding methyl ether are more potent inhibitors of cells and are completely resistant to degradation in vitro. At the same time, these cultures demonstrated the ability to inactivate a-amanitin. Implications regarding the selection of amatoxin-resistant lines in other plant species are made relative to these phenomena and their relation to polyphenoloxidase levels in plants. MATERIALS

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“…Chloroplast DNA-dependent RNA polymerase has been extensively purified from maize (1-3), pea (4), and spinach (5). Preparations of partially purified maize polymerase transcribe cloned homologous maize chloroplast DNA sequences in preference to bacterial vector sequences from supercoiled plasmids when a 27-kDa polypeptide, the S factor, is present (6).…”

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Binding and transcription of relaxed DNA templates by fractions of maize chloroplast extracts.

Zaitlin

Bogorad

1989

Proc. Natl. Acad. Sci. U.S.A.

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Preparations of partially purified chloroplast DNA-dependent RNA polymerase from maize and some other plants transcribe cloned chloroplast genes preferentially and much more actively from appropriately negatively supercoiled templates than from relaxed templates. We have found that the polymerase in such fractions does not bind to promoter regions of the maize chloroplast genes psbA and rbcL on small linear DNA fragments but that some protein(s) in unfractionated chloroplast extracts does bind. DEAE chromatography of the extracts has permitted the separation of a DNA-binding fraction from the bulk ofthe RNA polymerase activity. The binding fraction contains plastid RNA polymerase activity that is relatively independent of template topology.Chloroplast DNA-dependent RNA polymerase has been extensively purified from maize (1-3), pea (4), and spinach (5). Preparations of partially purified maize polymerase transcribe cloned homologous maize chloroplast DNA sequences in preference to bacterial vector sequences from supercoiled plasmids when a 27-kDa polypeptide, the S factor, is present (6). Such enzyme preparations transcribe cloned maize chloroplast genes rbcL (for the large subunit of ribulose-bisphosphate carboxylase) and atpBE (for the ,B and E subunits of the coupling factor for photophosphorylation) about 50 times more actively from optimally negatively supercoiled templates than from relaxed templates, but the relative transcription of the two genes from a single bacterial plasmid varies with negative superhelicity of the template (7,8). Transcription of cloned maize chloroplast genes rpS4 (for ribosomal protein S4) and psbA (for the QB protein of photosystem II) in vitro by maize plastid RNA polymerase preparations is also much greater from negatively supercoiled templates than from relaxed templates (9). Negative superhelicity of the template has been shown to affect transcription by RNA polymerase preparations from chloroplasts of other higher plant species as well (10, 11).Relative roles of transcription, postranscriptional processing, and translation in chloroplast differentiation are not well defined and appear to differ among plants. The abundance of transcripts of a single maize chloroplast gene can be substantially different in adjacent mesophyll and bundle-sheath cells and in etiolated vs. greening or green leaves (12-14); differences in transcription may contribute to such variations. Activity of maize chloroplast RNA polymerase increases substantially during light-induced greening of darkgrown maize although commensurate increases in major polypeptides are not seen (15).The present work was undertaken to examine interactions between cloned maize chloroplast gene sequences and maize chloroplast proteins. We have identified a region of the maize plastid gene psbA to which a protein (or proteins) in chloroplast extracts binds. We have also found that DEAE fractions of chloroplast extracts with the most DNA-binding material contain RNA polymerase activity that transcribes chloroplast genes fr...

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Chloroplast RNA polymerase from spinach: purification and DNA-binding proteins

Cited by 59 publications

References 21 publications

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

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

Sensitivity of Carrot Cell Cultures and RNA Polymerase II to Amatoxins

Binding and transcription of relaxed DNA templates by fractions of maize chloroplast extracts.

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