Specific in vitro transcription of 16S rRNA gene by pea chloroplast RNA polymerase

Sun, Eric I.; Shapiro, D R; Wu, B. W.; Tewari, Κ. Κ.

doi:10.1007/bf00027135

Cited by 34 publications

(21 citation statements)

References 19 publications

(20 reference statements)

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“…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.…”

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“…Chloroplasts were isolated from 250 g of young pea leaves and disrupted with 2.5% (final concentration) Triton X-100 as described previously (36). The Triton X-100-disrupted chloroplasts were first loaded onto a 100 ml of DEAE-cellulose (DE-52; Whatman Ltd., London, England) equilibrated in buffer A (10 mM Tris hydrochloride [pH 8.0], 50 mM 3-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 25% glycerol) with 0.1 M (NH4)2SO4.…”

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“…Enzymatic activity was assayed in a 0.1-ml mixture containing 500 ,uM ultrapure ATP, GTP, and CTP (Pharmacia, Inc., Piscataway, N.J.), 1 30 min, and acid-insoluble radioactivity was determined as described previously (36).…”

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In vitro analysis of the pea chloroplast 16S rRNA gene promoter.

Sun¹,

Wu²,

Tewari³

1989

Mol. Cell. Biol.

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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...

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In vitro analysis of the pea chloroplast 16S rRNA gene promoter.

Sun¹,

Wu²,

Tewari³

1989

Mol. Cell. Biol.

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“…1A). The 16S rRNA promoter contains prokaryotic -10 and -35 elements (Sun et al, 1986;Baeza et al, 1991) and therefore, it should be transcribed by a plastid-encoded RNA polymerase. According to Rajasekhar et al (1991), the highly purified RNA polymerase from pea chloroplasts can transcribe the 16S rRNA and other mRNAs in vitro.…”

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confidence: 99%

Cool Temperature-Induced Chlorosis in Rice Plants (II. Effects of Cool Temperature on the Expression of Plastid-Encoded Genes during Shoot Growth in Darkness)

1996

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It has been proposed that cool temperature-induced chlorosis (CTIC) in / d i c a cultivars of rice (Oryza sativa L.) is caused by cell growth and plastid development being impeded at cool temperatures. Since it is well known that the overall rate of transcription of plastid-encoded genes changes dramatically during the early phases of plastid development, in this study we focused on the patterns of expression of these genes. Northern blot analysis revealed that the level of 16s rRNA is decreased in a CTIC-sensitive rice cultivar grown at a cool temperature. The expression of the gene for the p subunit of plasmid RNA polymerase (rpot?) was shown to be somewhat disturbed, particularly in terms of its resuppression under cool conditions. The level of transcripts or proteins of plastid-encoded photosynthetic genes was also decreased in a CTIC-sensitive cultivar at a cool temperature. These results suggest that the temperature-dependent inhibition of the onset of gene expression encoding the transcription/translation apparatus may be primarily involved in the mechanism causing CTIC.CTIC in developing seedlings is one symptom of cold injury in rice plants (Ovyza sativa L.), particularly in cultivars of the Indica type (Choung and Omura, 1982;Kaimori and Takahashi, 1985). When seedlings are exposed to cool weather, the newly emerging leaves lack pigments. Once such chlorotic leaves have developed, they remain white even at permissive temperatures without withering. To gain insight into the molecular mechanism of CTIC in rice plants, we previously established a model using darkgrown seedlings to mimic in the laboratory the CTIC that occurs in the field (Yoshida et al., 1996). In our model, the growth of immature leaves and the development of undifferentiated plastids to mature etioplasts in darkness was influenced by cool temperatures.The expression of CTIC in SUR is bimodally dependent on temperature, suggesting a growth-dependent phenomenon (Yoshida et al., 1996). Biochemical and ultrastructural studies suggest that a reduction in the level of NADPHprotochlorophyllide oxidoreductase and arrested etioplast development might be closely linked to the expression of the CTIC phenotype.In monocots, proplastids are converted into mature organelles during the development of mesophyll leaf cells * Corresponding author; e-mail riichi@bansui.ige.tohoku.ac.jp; fax 181-22-263-9845. from leaf primordial cells (Baumgatner et al., 1989). During the early phases of development the overall rate of transcription of plastid-encoded genes changes dramatically (Baumgartner et al., 1993). In particular, the rate of transcription and the level of transcripts of genes encoding the transcription/ translation apparatus in plastids are dramatically elevated relative to most of the genes that encode the photosynthetic apparatus early in the development of organelles in synchrony with cell growth (Bisanz-Seyer et al., 1989;Baumgartner et al., 1993;Harrak et al., 1995). The rate of posttranscriptional processes, including RNA processing and stabili...

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“…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).…”

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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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Specific in vitro transcription of 16S rRNA gene by pea chloroplast RNA polymerase

Cited by 34 publications

References 19 publications

In vitro analysis of the pea chloroplast 16S rRNA gene promoter.

In vitro analysis of the pea chloroplast 16S rRNA gene promoter.

Cool Temperature-Induced Chlorosis in Rice Plants (II. Effects of Cool Temperature on the Expression of Plastid-Encoded Genes during Shoot Growth in Darkness)

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

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