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

Hanley‐Bowdoin, Linda; Orozco, Emil M.; Chua, Nam‐Hai

doi:10.1128/mcb.5.10.2733

Cited by 53 publications

(31 citation statements)

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“…A and by 5' processing in higher-plant chloroplasts. For example, rbcL and psbB mRNAs each have two 5' ends, one of which is generated by processing (8,10,21,32). Spinach atpB mRNA has five 5' termini, four of which result from alternative initiation sites.…”

Section: Resultsmentioning

confidence: 99%

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

Sakamoto¹,

Sturm²,

Kindle³

et al. 1994

Mol. Cell. Biol.

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Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes. In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons. By generating specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b6lf complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription ofpetD originates from the upstream petA promoter. The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants. The location and function of the processing site were further examined by inserting petD-uidAl fusion genes into the chloroplast genome (uid4 is an Escherichia coli gene that encodes ,-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uid4 transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end. When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was inserted into the same site, a dicistronic petA-uidA4 transcript accumulated but no monocistronic uid4 transcript could be detected, suggesting that a processing site lies at least partially within the region from -1 to +25. ,1-Glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uid4 transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation. One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages.Higher-plant chloroplast genes are organized as operons that are transcribed into polycistronic primary transcripts. Splicing and other endo-and exonucleolytic cleavages subsequently occur to generate mature transcripts (1,9,12,32). One such operon in higher plants contains the petD gene, which encodes subunit IV of the cytochrome bdlf complex. Its genomic location is conserved in higher plants (13,30) and cyanobacteria (15); however, its mode of expression varies. In spinach chloroplasts, a primary transcript containing the psbB, psbH, petB, and petD coding regions is processed into 18 RNA species, including monocistronicpsbB andpsbH transcripts and a dicistronic petB-petD transcript (32). As no monocistronic petD mRNA has been detected in spinach chloroplasts, the coding region is presumed to be translated from the dicistronic message. The transcription ofpetD RNA in maize chloroplasts also occurs as part of the psbB operon, but the major translatable form of the petD message is monocistronic. Nonetheless, polycistronic transcripts can be immunoprecipitated from maize polysomes with subunit IV-spe...

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Section: Resultsmentioning

confidence: 99%

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

Sakamoto¹,

Sturm²,

Kindle³

et al. 1994

Mol. Cell. Biol.

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“…The' -180' position of the rbcL gene is the site of transcription initiation in vivo [22] and in vitro [24]. The' -65' rbcL transcript is presumably an RNA processing product, as in maize [ 14]. Four in vivo transcripts for the atpB gene were previously identified, containing either 454, 274, 179 or 99 nucleotides of untranslated leader sequence [22].…”

Section: In Vitro Transcriptionmentioning

confidence: 99%

“…Part of this heterogeneity in RNA is due to posttranscriptional RNA processing events [14,30,2]. However, some chloroplast genes such as the spinach gene encoding the fl subunit of the ATPase (atpB) were thought to contain multiple promoters [22].…”

Section: Introductionmentioning

confidence: 99%

“…By transcribing plasmid DNAs containing putative chloroplast promoters 5' to the threonine attenuator, it is possible to rapidly assay for promoter activity in vitro. However, RNAs produced in vitro by a crude chloroplast transcription extract can also result from RNA processing activity [14]. Because chloroplast and E. coli promoters are similar in sequence, transcription of suspected chloroplast promoters by E. coli RNA polymerase is a useful means to distinguish between production of primary versus processed RNAs.…”

Section: Introductionmentioning

confidence: 99%

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A primary transcript in spinach chloroplasts that completely lacks a 5′ untranslated leader region

et al. 1990

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A fifth, previously undetected transcript for the plastid gene encoding the beta subunit of spinach chloroplast ATPase (atpB) has recently been identified in vitro. In this report we show that this transcript is present in vivo and its 5' end is at the translation start codon. When synthesized in vitro using spinach chloroplast RNA polymerase, the 5' end of this novel transcript is located at the beginning of the second codon of the atpB coding sequence. Although this atpB transcript lacks an untranslated leader region, it is an abundant RNA in vivo and is associated with crude polysomal fractions, as are the four larger atpB transcripts. In vitro synthesis of the leaderless transcript does not occur for a plasmid DNA in which the "-35' region of the promoter is deleted.

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“…Plasmid DNA used throughout this study is pZmc812 (provided by L. Hanley-Bowdoin of our laboratory) which is a pUC13A sub-clone containing a Sau3A-BamH1 fragment of the maize chloroplast DNA insert in pZmc810 (6). The construct contains only the -300 site previously characterized as the transcription initiation site in vivo and in vitro (6,10).…”

Section: Preparation Of Chloroplast Extracts and Plasmid Templatesmentioning

confidence: 99%

Chloroplast DNA gyrase and in vitro regulation of transcription by template topology and novobiocin

Lam

Chua

1987

Plant Mol Biol

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We have examined the effects of novobiocin and template topology on the transcription of two chloroplast genes encoding the large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) and the beta subunit of the chloroplast ATPase (atpB), in an in vitro transcription system. The template topology was monitored by agarose gel electrophoresis while the in vitro transcripts were determined by 5' S1 nuclease analysis under identical conditions. We discovered that our chloroplast transcription extracts contain a DNA gyrase activity and a chromatographically separable topoisomerase I activity. Incubation of a supercoiled template with the extracts under the same conditions in which transcription assays were carried out leads to a decrease in the supercoiled from and concomitant appearance of distinct topoisomers. More extensive relaxation of the supercoiled template occurs when nucleotide triphosphates are omitted from the reaction mixture or when a low concentration (25 μg/ml) of novobiocin is added. Higher concentrations (≥ 250 μg/ml) of the drug, however, also inhibit the topoisomerase I activity. The transcription of the atpB gene is inhibited by lower concentrations of novobiocin as compared to the rbcL gene in the same reaction mixture. Relaxed, closed circular template and linearized DNA are not substrates for chloroplast transcription extracts, although they are transcribed accurately by the E. coli RNA polymerase under our conditions. Control of in vitro transcription of the two chloroplast genes by template topology can also be demonstrated by modulating the relative activity for the topoisomerases in the transcription extract. Our results suggest that changes in template topology may be a mechanism by which chloroplast genes are differentially regulated and the chloroplast DNA gyrase and topoisomerase I are key enzymes for this mode of regulation in vivo.

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

Cited by 53 publications

References 50 publications

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

A primary transcript in spinach chloroplasts that completely lacks a 5′ untranslated leader region

Chloroplast DNA gyrase and in vitro regulation of transcription by template topology and novobiocin

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