Selective in vitro transcription of chloroplast genes

Gruissem, Wilhelm; Narita, J O; Greenberg, Bruce M.; Prescott, David M.; Hallick, Richard B.

doi:10.1002/jcb.240220104

Cited by 50 publications

(31 citation statements)

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“…However, evidence supporting this hypothesis is indirect, based on DNA sequence homology (60) and expression of chloroplast genes in bacterial systems (20,22,24,52). Only a few studies have examined directly the synthesis of chloroplast RNAs at the molecular level by using plastid enzymes in vitro (16,27,28,34,45). In the work reported here, we used homologous and heterologous chloroplast enzymes in vitro to characterize two aspects of maize plastid RNA synthesis, i.e., the initiation of rbcL gene transcription and 5'-end processing of the primary transcript.…”

Section: Discussionmentioning

confidence: 99%

In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

Hanley‐Bowdoin¹,

Orozco²,

Chua³

1985

Mol. Cell. Biol.

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The large subunit gene (rbcL) of ribulose 1,5-bisphosphate carboxylase was transcribed in vitro by using maize and pea chloroplast extracts and a cloned plastid DNA template containing 172 base pairs (bp) of the maize rbcL protein-coding region and 791 bp of upstream sequences. Three major in vitro RNA species were synthesized which correspond to in vivo maize rbcL RNAs with 5' termini positioned 300, 100 to 105, and 63 nucleotides upstream of the protein-coding region. A deletion of 109 bp, including the "-300" 5' end (the 5' end at position -300), depressed all rbcL transcription in vitro. A plasmid DNA containing this 109-bp fragment was sufficient to direct correct transcription initiation in vitro. A cloned template, containing 191 bp of plastid DNA which includes the -105 and -63 rbcL termini, did not support transcription in vitro. Exogenously added -300 RNA could be converted to the -63 transcript by maize chloroplast extract. These results established that the -300 RNA is the primary maize rbcL transcript, the -63 RNA is a processed form of the -300 transcript, and synthesis of the -105 RNA is dependent on the -300 region. The promoter for the maize rbcL gene is located within the 109 bp flanking the -300 site. Mutagenesis of the 109-bp chloroplast sequence 11 bp upstream of the -300 transcription initiation site reduced rbcL promoter activity in vitro.Ribulose 1,5-bisphosphate carboxylase (RUBISCO) catalyzes CO2 fixation, the first step of the Calvin cycle (40). In higher plants, the chloroplast enzyme is composed of eight identical, catalytic polypeptides of 50,000 to 60,000 kilodaltons and eight smaller polypeptides of 12,000 to 20,000 kilodaltons (2). The small subunit, whose function is unknown, is encoded in the nucleus by a small multigene family (7,14,19). The large subunit gene (rbcL) is present as a single copy on the multicopy chloroplast genome (5). Under most conditions, the synthesis of the two RUBISCO subunits is coordinated (55) and regulated by light (15, 50) and cytokinins (23). In maize and other C4 plants, RUBISCO expression is leaf cell-type specific (30). The maize mRNAs encoding the two subunits are present in bundle sheath cells but not in mesophyll cells, which assimilate CO2 via ribulose 1,5-bisphosphate and C4 dicarboxylic acids, respectively (11,35).In chloroplast genomes of higher plants, the genes for the large subunit of RUBISCO and the beta subunit of ATP synthetase (atpB) are adjacent and transcribed divergently (31, 60). The 5' ends of the two genes are separated by approximately 150 base pairs (bp) (52). Northern hybridization analyses of transcripts from a broad spectrum of angiosperms indicate that the rbcL gene is generally transcribed as a 1.6-kilobase mRNA, although larger transcripts are also observed (46). Si nuclease protection experiments have revealed the presence of two 5' termini for the rbcL RNAs of maize, barley, spinach, and peas (16,21,41,49,62.) Tobacco has a single rbcL RNA with its 5' end at position -180 relative to the coding region (53). In maize ...

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Section: Discussionmentioning

confidence: 99%

In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

Hanley‐Bowdoin¹,

Orozco²,

Chua³

1985

Mol. Cell. Biol.

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2005

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Transcription in plastids is mediated by a plastid-encoded multimeric (PEP) and a nuclear-encoded single-subunit RNA polymerase (NEP) and a still unknown number of nuclear-encoded factors. By combining gel filtration and affinity chromatography purification steps, we isolated transcriptionally active chromosomes from Arabidopsis thaliana and mustard (Sinapis alba) chloroplasts and identified 35 components by electrospray ionization ion trap tandem mass spectrometry. Eighteen components, called plastid transcriptionally active chromosome proteins (pTACs), have not yet been described. T-DNA insertions in three corresponding genes, ptac2, -6, and -12, are lethal without exogenous carbon sources. Expression patterns of the plastid-encoded genes in the corresponding knockout lines resemble those of Drpo mutants. For instance, expression of plastid genes with PEP promoters is downregulated, while expression of genes with NEP promoters is either not affected or upregulated in the mutants. All three components might also be involved in posttranscriptional processes, such as RNA processing and/or mRNA stability. Thus, pTAC2, -6, and -12 are clearly involved in plastid gene expression.

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“…The extent of clustering of tRNA genes is highest in Euglena chloroplast DNA. For Euglena, spinach (Spinacea oleracea) and pea (Pisum sativum) chloroplasts, two different transcription systems have been described [11,12,14,15,20]. The TAC (transcriptionally active chromosome) RNA polymerase activity is tightly bound to chloroplast DNA and transcribes rRNA genes [4,5,10,19].…”

Section: Introductionmentioning

confidence: 99%

“…The TAC (transcriptionally active chromosome) RNA polymerase activity is tightly bound to chloroplast DNA and transcribes rRNA genes [4,5,10,19]. The soluble RNA polymerase extract is active in tRNA and mRNA transcription and tRNA processing [9,10,15,27]. Enzymes involved in post-transcriptional maturation of chloroplast pre-tRNAs have been identified, but not purified.…”

Section: Introductionmentioning

confidence: 99%

Processing of mono-, di- and tricistronic transfer RNAs precursors in a spinach or pea chloroplast soluble extract

et al. 1988

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Monomeric, dimeric and trimeric chloroplast tRNA precursors from Euglena gracilis were synthesized by Sp6, T7 or T3 RNA polymerases using an in vitro transcription system. The length of the 3' and 5' ends of these precursors was varied to facilitate the identification of processing intermediates, and to study the effect of the structure of the tRNA precursors on the processing reactions. All the tRNA precursors studied, independent of their structure, are processed to mature tRNAs in both spinach and pea chloroplast soluble extracts. 5'-and 3' endonucleases are involved in the cleavage of 5' and 3' ends of the pre-tRNAs. These two reactions are not ordered in vitro. Other enzymatic activities can be detected in the chloroplast soluble extract including exonucleases, and CCA-adding enzyme.

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Selective in vitro transcription of chloroplast genes

Cited by 50 publications

References 31 publications

In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

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

Processing of mono-, di- and tricistronic transfer RNAs precursors in a spinach or pea chloroplast soluble extract

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